Intraflagellar transport

ABSTRACT

The invention relates to various intraflagellar transport (IFT) polypeptides and the nucleic acids that encode them. The new IFT particle polypeptides and nucleic acids can be used in a variety of diagnostic, screening, and therapeutic methods.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/206,923, filed on May 24, 2000, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This invention relates to intraflagellar transport proteins.

BACKGROUND

[0003] Cilia are tiny cellular structures that protrude from cells. Theyare about 0.25 micrometers in diameter and contain a bundle ofmicrotubules. They are widespread among living organisms, occurring inmost animals, many single-celled eukaryotes, and in some lower plants.

[0004] Cilia tend to function in one of two ways. They may move fluidacross the surface of the cell or they may propel cells through a fluid.They may also serve to gather food. In humans, cilia on the surfaces ofrespiratory epithelia function to push mucus and trapped particles anddead cells out of the lungs. Cilia also function to carry eggs throughthe oviduct. Cilia function in myriad ways in different kinds of cells.

[0005] Flagella are structures related to cilia. They are similar ininternal structure but tend to be much longer than cilia. Sperm cellsare propelled by flagella, as are many other single-celled eukaryotes.

[0006] Groups of cilia tend to move together in coordinatedunidirectional waves. The motion made by each individual cilium iswhiplike. This motion includes two phases. First, the cilium extendsforward, pushing against the surrounding liquid as it goes. At the endof its forward stroke, the cilium bends, reducing viscous drag as itpulls itself back to its original position. By contrast, flagella tendto propagate quasi-sinusoidal waves. Despite the differences in theirexternal motions, the molecular basis of movement in both cilia andflagella appear to be the same.

[0007] Cilia and flagella move by bending their core—the axoneme. Theaxoneme is composed of microtubules and associated proteins. The patternof microtubules is distinctive: nine pairs of microtubules that form aring around two single microtubules. This arrangement is typicallyreferred to as “9 +2”. The pairs are composed of one complete and onepartial microtubule. These microtubules extend the full length of theaxoneme, which can range in length from 10200 micrometers.

[0008] Intraflagellar transport (IFT) is a dynein and kinesin-basedmotility process in which nonmembrane-bound particles move alongflagellar microtubules, just beneath the flagellar membrane, from thebase to the tip of the flagellum and back. IFT is essential for theassembly and maintenance of all cilia and flagella, including non-motileprimary cilia and sensory cilia. Recent results indicate that defects inIFT are a primary cause of several human diseases.

SUMMARY

[0009] The invention is based on the discovery of various new IFTparticle polypeptides and the genes that encode them.

[0010] In general, the invention features, an isolated nucleic acidmolecule selected from the group consisting of: a) a nucleic acidmolecule having a nucleotide sequence which is at least 90% identical tothe nucleotide sequence of Chlamydomonas intraflagellar transport (IFT)particle protein gene 20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2, ora complement thereof; b) a nucleic acid molecule comprising at least 15nucleotide residues and having a nucleotide sequence identical to atleast 15 consecutive nucleotide residues of the nucleotide sequence ofChlamydomonas IFT particle protein gene 20, 27, 46, 52, 57, 72, 88, 122,or 139, or Che-2, or a complement thereof; c) a nucleic acid moleculewhich encodes a polypeptide comprising the amino acid sequence ofChlamydomonas IFT particle protein 20, 27, 46, 52, 57, 72, 88, 122, 139,or Che-2; or d) a nucleic acid molecule which encodes a polypeptidecomprising at least 10 amino acids and having an amino acid sequenceidentical to at least 10 consecutive amino acids of the amino acidsequence of Chlamydomonas IFT particle protein 20, 27, 46, 52, 57, 72,88, 122, 139, or Che-2.

[0011] The nucleic acid molecules can further include nucleic acidsequences encoding a heterologous polypeptide. The invention alsofeatures a vector including the nucleic acid molecules and host cellsincluding the new nucleic acid molecules, such as non-human mammalianhost cells.

[0012] The invention also features an isolated polypeptide selected fromthe group consisting of: a) a polypeptide comprising at least 10 aminoacids and having an amino acid sequence identical to at least 10consecutive amino acids of the amino acid sequence of Chlamydomonasintraflagellar transport (IFT) particle protein 20, 27, 46, 52, 57, 72,88, 122, 139, or Che-2; b) a polypeptide comprising the amino acidsequence of Chlamydomonas IFT particle protein 20, 27, 46, 52, 57, 72,88, 122, 139, or Che-2, wherein the polypeptide comprises one or moreconservative amino acid substitutions that do not inhibit the biologicalactivity of the polypeptide relative to a corresponding nativeChlamydomonas IFT particle protein; and c) a polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 90% identical to a nucleic acid consisting of thenucleotide sequence of Chlamydomonas IFT particle protein gene 20, 27,46, 52, 57, 72, 88, 122, 139, or Che-2, or a complement thereof.

[0013] In another aspect, the invention features an antibody thatselectively binds to the new polypeptides.

[0014] Yet other nucleic acid molecules of the invention include thosehaving sequences that (1) are at least 90% identical to the nucleotidesequence of mouse intraflagellar transport (IFT) particle protein gene57 (or are complements thereof); (2) are at least 15 nucleotide residueslong and have a sequence identical to at least 15 consecutive nucleotideresidues of the nucleotide sequence of mouse IFT particle protein gene57 (or complements thereof); (3) encode a polypeptide that is or thatincludes the amino acid sequence of mouse IFT particle protein 57; or(4) encode a polypeptide having at least 10 amino acids and an aminoacid sequence identical to at least 10 consecutive amino acids of theamino acid sequence of mouse IFT particle protein 57. For example, anucleic acid molecule of the invention can be a nucleic acid having thenucleotide sequence of mouse IFT particle protein gene 57 (or acomplement thereof) or a nucleic acid molecule that encodes apolypeptide having the amino acid sequence of mouse IFT particle protein57.

[0015] Yet other polypeptides of the invention include those havingsequences that (1) include at least 10 amino acid residues and have anamino acid sequence identical to at least 10 consecutive amino acids ofthe amino acid sequence of mouse intraflagellar transport (IFT) particleprotein 57; (2) include the amino acid sequence of mouse IFT particleprotein 57; or (3) are encoded by a nucleic acid molecule having anucleotide sequence that is at least 90% identical to a nucleic acidconsisting of the nucleotide sequence of mouse IFT particle protein gene57 (or a complement thereof). For example, a polypeptide of theinvention can include the amino acid sequence of mouse IFT particleprotein 57. Any of the polypeptides described herein can include one ormore conservative amino acid substitutions that do not inhibit thebiological activity of the polypeptide relative to native mouse IFTparticle protein 57 (e.g., polypeptides that retain at least 50% (e.g.60%, 75%, 80%, 90%, or 95% or more) of one or more of the biologicalactivities of a native IFT polypeptide (e.g. particle protein 57)).

[0016] Various methods are also within the scope of the invention. Forexample, the invention features a method for identifying a candidatecompound that modulates (e.g., inhibits or stimulates) the activity ofmouse intraflagellar transport (IFT) particle protein 57. The method canbe carried out, for example, by contacting a test compound with anisolated IFT particle polypeptide and determining whether the testcompound interacts with the polypeptide. Interaction indicates that thetest compound is a candidate modulator of mouse IFT particle protein 57.Similarly, one can carry out methods to identify a candidate compoundthat modulates (e.g., inhibits or stimulates) the activity of a humanintraflagellar transport (IFT) particle protein. These methods can becarried out, for example, by contacting a test compound with an isolatedIFT particle polypeptide and determining whether the test compoundinteracts with the polypeptide. Here again, interaction indicates thatthe test compound is a candidate modulator of a human IFT particleprotein. The isolated human IFT particle polypeptide can be human IFTparticle polypeptide 20-1, 20-2, 20-3, 27, 46, 52, 57-1, 57-2, 72, 88,122, 139-1, 139-2 or Che-2.

[0017] The methods described above can include additional steps. Forexample, one can contact that candidate modulator with one or more cells(e.g. cultured cells) that have functional cilia and determine whetherthe modulator modulates (e.g. inhibits or stimulates) cilia function. Inthe event cilia function is inhibited, the candidate modulate is an IFTparticle protein inhibitory agent. In other embodiments, the candidatemodulator can be contacted with one or more cells (e.g. cultured cells)that have non-functional or functionally impaired cilia (e.g. a cell orcells lacking a specific IFT particle protein). Restoration (partial orcomplete) of cilia function indicates that the candidate modulator is anIFT particle protein restorative agent.

[0018] Other methods of the invention can be used to identify acandidate compound that restores the activity of a defective or absenthuman intraflagellar transport (IFT) particle protein. These methods canbe carried out by, for example, obtaining a mixture of isolated IFTparticle polypeptides that include (i) all but one of the IFT particlepolypeptides required to form the IFT particle, and (ii) a medium thatenables the IFT particle polypeptides to form the IFT particle when allnormal IFT particle polypeptides that constitute that IFT particle arepresent. The mixture is then contacted with a test compound, after whichone determines whether the test compound enables the IFT particle to beformed. IFT particle formation indicates that the test compound is acandidate compound that restores the activity of a defective or absenthuman IFT particle protein. These methods can be carried out bycontacting the candidate compound with one or more cells (e.g., culturedcells) that have non-functional (or impaired) cilia and that lack aspecific IFT particle protein. One can then determine whether thecandidate compound restores cilia function, which would indicate thatthe candidate compound is an IFT particle protein restorative agent.These methods (and others of the invention) can be carried out with ahuman IFT particle polypeptide (e.g., human IFT particle polypeptides20-1, 20-2, 20-3, 27, 46, 52, 57-1, 57-2, 72, 88, 122, 139-1, 139-2 orChe-2; or combinations thereof).

[0019] Diagnostic methods are also within the scope of the invention.For example, the invention features a method of diagnosing a disorder ina tissue in a subject that is associated with (e.g. caused by, in wholeor in part) a defective or absent human intraflagellar transport (IFT)particle protein. The method can be carried out, for example, bydisrupting cells from a tissue sample, contacting the disrupted cellswith an antibody that specifically binds to a normal human IFT particleprotein, and detecting binding of the antibody to any IFT particleprotein in the sample. An absence of specific binding indicates that thetissue is one in which IFT particle protein is defective or absent.Disorders (or diseases or conditions) associated with this tissue defectinclude, but are not limited to, kidney disease, retinal disorders,thyroid disorders, chondrocyte disease, olfactory disease, azoospermia,and primary ciliary dyskinesia.

[0020] In other embodiments, the methods are methods of treatment. Forexample, the invention features a method of treating a disorder in asubject (e.g. a disorder associated with a defective or absentintraflagellar transport (IFT) protein) by administering to the subjecta human IFT particle polypeptide. The amount of the polypeptide is anamount that is effective to compensate for the defective or absent IFTparticle protein. Rather than administering a polypeptide of theinvention, one can administer a nucleic acid molecule that encodes it.For example, one can administer a human IFT particle polypeptide (e.g,human IFT particle polypeptides 20-1, 20-2, 20-3, 27, 46, 52, 57-1,57-2, 72, 88, 122, 139-1, 139-2 or Che-2) or a nucleic acid moleculeencoding such a polypeptide. The polypeptides or nucleic acid moleculescan be delivered with a pharmaceutically acceptable carrier, excipient,or diluent.

[0021] The methods of the invention can also be carried out to treat aninfection in a subject that is caused by a pathogen (e.g a nematode,bacteria, protozoa or insect) that has an intraflagellar transport (IFT)particle protein. The subjects are treated by administering to them aneffective amount of an agent that inhibits the function of the IFTparticle protein (e.g., an antibody that binds specifically to the IFTparticle protein). The diagnostic and therapeutic methods of theinvention can be used to diagnose or treat mammals (e.g. humans or otherprimates, or domesticated or farm animals) as well as plants.

[0022] Another suitable method for identifying compounds that inhibit orrestore IFT function involves screening for small molecules thatspecifically bind to IFT proteins. A variety of suitable binding assaysare known in the art as described, for example, in U.S. Pat. Nos.5,585,277 and 5,679,582, incorporated herein by reference. For example,in various conventional assays, test compounds can be assayed for theirability to bind to a polypeptide by measuring the ability of the smallmolecule to stabilize the polypeptide in its folded, rather thanunfolded, state. More specifically, one can measure the degree ofprotection against unfolding that is afforded by the test compound. Testcompounds that bind to an IFT protein with high affinity cause, forexample, a significant shift in the temperature at which the polypeptideis denatured. Test compounds that stabilize the polypeptide in a foldedstate can be further tested for IFT inhibitive or restorative activityin a standard susceptibility assay.

[0023] The IFT polypeptides also can be used in assays to identify testcompounds that bind to the polypeptides. Test compounds that bind to IFTpolypeptides then can be tested, in conventional assays, for theirability to inhibit or restore IFT function. Test compounds that bind toIFT polypeptides are candidate IFT inhibitive or restorative agents, incontrast to compounds that do not bind to IFT polypeptides. As describedherein, any of a variety of art-known methods can be used to assay forbinding of test compounds to IFT polypeptides. If desired, the testcompound can be immobilized on a substrate, and binding of the testcompound to an IFT polypeptide is detected as immobilization of an IFTpolypeptide on the immobilized test compound. Immobilization of an IFTpolypeptide on the test compound can be detected in an immunoassay withan antibody that specifically binds to an IFT polypeptide.

[0024] Also included in the invention is a method for identifying acandidate IFT restorative agent useful for treating abnormal IFTfunction by: (a) contacting a polypeptide encoded by an IFT nucleic acidwith a test compound; and (b) detecting binding of the test compound tothe polypeptide, wherein a compound that binds to the IFT polypeptideindicates that the compound is a candidate IFT function restorativeagent, and wherein the polypeptide is encoded by a gene selected fromthe group consisting of a first nucleic acid molecule which encodes apolypeptide containing the amino acid sequence of a polypeptide of theinvention or a naturally occurring allelic variant of a polypeptide ofthe invention, wherein the first nucleic acid molecule hybridizes to asecond nucleic acid molecule under stringent conditions. The method canfurther include a step of determining whether the candidate compoundthat binds to the IFT polypeptide inhibits growth of cells or organisms,relative to growth of cells or organisms grown in the absence of thetest compound, wherein inhibition of growth indicates that the candidatecompound is an anti-IFT agent.

[0025] In one example, the test compound is immobilized on a substrate,and binding of the test compound to the IFT polypeptide is detected asimmobilization of the IFT polypeptide on the immobilized test compound.Immobilization of the IFT polypeptide on the test compound can bedetected in an immunoassay with an antibody that specifically binds tothe IFT polypeptide.

[0026] In one example, the test compound is selected from the groupconsisting of polypeptides, ribonucleic acids, small molecules (e.g.,organic or inorganic), aptamers, peptidomimetics, carbohydrates, anddeoxyribonucleic acids.

[0027] By “operably linked” is meant that a gene and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

[0028] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0029] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0030]FIG. 1 is a representation of a model of the IFT particlesinvolved in intraflagellar transport towards and away from the tip of aflagellum.

[0031]FIG. 2 is a representation of a large scale model of the IFTparticles involved in intraflagellar transport towards and away from thetip of the flagellum.

[0032]FIG. 3 is a representation of the flagellar pore complex.

[0033]FIG. 4 is a representation of a model of how flagellar proteinsare targeted to the flagellum.

[0034]FIG. 5 is a representation of a model of the photoreceptor cellIFT.

[0035]FIG. 6A is a representation of Chlamydomonas IFT20 particlepolypeptide (SEQ ID NO:2).

[0036]FIG. 6B is a representation of Chlamydomonas IFT20 particlenucleic acid (SEQ ID NO: 1).

[0037]FIG. 6C is a representation of human IFT20 particle polypeptide(SEQ ID NO:23).

[0038]FIG. 6D is a representation of human IFT20 particle polypeptide(SEQ ID NO:24).

[0039]FIG. 6E is a representation of human IFT20 particle polypeptide(SEQ ID NO:25).

[0040]FIG. 7A is a representation of Chlamydomonas IFT27 particlepolypeptide (SEQ ID NO:4).

[0041]FIG. 7B is a representation of Chlamydomonas IFT27 particlenucleic acid (SEQ ID NO:3).

[0042]FIG. 7C is a representation of human IFT27 particle polypeptide(SEQ ID NO:26).

[0043]FIG. 8A is a representation of Chlamydomonas IF T46 particlepolypeptide (SEQ ID NO:6).

[0044]FIG. 8B is a representation of Chlamydomonas IFT46 particlenucleic acid (SEQ ID NO:5).

[0045]FIG. 8C is a representation of human IFT46 particle polypeptide(SEQ ID NO:27).

[0046]FIG. 9A is a representation of Chlamydomonas IFT52 particlepolypeptide (SEQ ID NO:8).

[0047]FIG. 9B is a representation of Chlamydomonas IFT52 particlenucleic acid (SEQ ID NO:7).

[0048]FIG. 9C is a representation of human IFT52 particle polypeptide(SEQ ID NO:28).

[0049]FIG. 9D is a representation of Caenorhabditis elegans IFT52particle polypeptide (SEQ ID NO:29).

[0050]FIG. 10A is a representation of Chlamydomonas IFT57 particlepolypeptide (SEQ ID NO:10).

[0051]FIG. 10B is a representation of Chlamydomonas IFT57 particlenucleic acid (SEQ ID NO:9).

[0052]FIG. 10C is a representation of mouse IFT57 particle polypeptide(SEQ ID NO: 12).

[0053] FIG 10D is a representation of mouse IFT57 particle nucleic acid(SEQ ID NO: 11).

[0054]FIG. 10E is a representation of human IFT57 particle polypeptide(SEQ ID NO:30).

[0055]FIG. 10F is a representation of human IFT57 particle polypeptide(SEQ ID NO:31).

[0056]FIG. 10G is a representation of Caenorhabditis elegans IFT57particle polypeptide (SEQ ID NO:32).

[0057]FIG. 11A is a representation of Chlamydomonas IFT72 particlepolypeptide (SEQ ID NO: 14).

[0058]FIG. 11B is a representation of Chlamydomonas IFT72 particlenucleic acid (SEQ ID NO:13).

[0059]FIG. 11C is a representation of human IFT72 particle polypeptide(SEQ ID NO:33).

[0060]FIG. 12A is a representation of Chlamydomonas IFT88 particlepolypeptide (SEQ ID NO:16).

[0061]FIG. 12B is a representation of Chlamydomonas IFT88 particlenucleic acid (SEQ ID NO:15).

[0062]FIG. 12C is a representation of human IFT88 particle polypeptide(SEQ ID NO:34).

[0063]FIG. 12D is a representation of Caenorhabditis elegans IFT88particle polypeptide (SEQ ID NO:35).

[0064]FIG. 13A is a representation of Chlamydomonas IFT122 particlepolypeptide (SEQ ID NO:18).

[0065]FIG. 13B is a representation of Chlamydomonas IFT122 particlenucleic acid (SEQ ID NO:17).

[0066]FIG. 13C is a representation of human IFT122 particle polypeptide(SEQ ID NO:36).

[0067]FIG. 13D is a representation of Caenorhabditis elegans IFT122particle polypeptide (SEQ ID NO:37).

[0068]FIG. 14A is a representation of Chlamydomonas IFT139 particlepolypeptide (SEQ ID NO:20).

[0069]FIG. 14B is a representation of Chlamydomonas IFT139 particlenucleic acid (SEQ ID NO:19).

[0070]FIG. 14C is a representation of human IFT139 particle polypeptide(SEQ ID NO:38).

[0071]FIG. 14D is a representation of human IFT139 particle polypeptide(SEQ ID NO:39).

[0072]FIG. 14E is a representation of Caenorhabditis elegans IFT139particle polypeptide (SEQ ID NO:40).

[0073]FIG. 15A is a representation of Chlamydomonas Che-2 polypeptide(SEQ ID NO:22).

[0074]FIG. 15B is a representation of Chlamydomonas Che-2 nucleic acid(SEQ ID NO:21).

[0075]FIG. 15C is a representation of human Che-2 polypeptide (SEQ IDNO:41).

[0076]FIG. 15D is a representation of Caenorhabditis elegans Che-2polypeptide (SEQ ID NO:42).

DETAILED DESCRIPTION

[0077] A. Intraflagellar Transport Intraflagellar transport (IFT) is adynein and kinesin-based motility process in which nonmembrane-boundparticles move along flagellar microtubules, just beneath the flagellarmembrane, from the base to the tip of the flagellum and back (see FIG.1). IFT is essential for the assembly and maintenance of all cilia andflagella, including non-motile primary cilia and sensory cilia. Recentresults indicate that defects in IFT are a primary cause of severalhuman diseases.

[0078] Much of our knowledge of the protein machinery and basic biologyof IFT has come from studies of the bi-flagellate alga Chlamydomonas,which is an excellent model system for biochemical and molecular geneticanalyses of proteins and processes occurring in the flagellum. IFT wasfirst observed in Chlamydomonas using Differential Interference Contrast(DIC) microscopy (Kozminski et al., Proc. Nat. Acad. Sci. U.S.A.,90:5519-5523, 1993), where particles were seen to move anterogradely(i.e., from base to tip) along the flagellum at ˜2.5 mm/s without pause,while apparently smaller particles moved retrogradely at ˜4 mm/s.Correlative light and electron microscopy revealed that the particlesbeing moved during IFT were organized into linear arrays termed “rafts”(Kozminski et al., Proc. Nat. Acad. Sci. U.S.A., 90:5519-5523, 1993).Electron microscopy of the arrays showed that they were connected byperiodic links to both the flagellar membrane and the B-tubule of theouter doublet microtubule (Kozminski et al., Proc. Nat. Acad. Sci.U.S.A., 90:5519-5523, 1993; Kozminski et al., J. Cell Biol.,131:1517-1527, 1995; Pazour et al., J. Cell Biol, 141:979-992, 1998).Biochemical characterization of the Chlamydomonas IFT particles revealedthat they are composed of at least 16 different polypeptides (Pipemo andMead, Proc. Natl. Acad. Sci., 94:4457-4462, 1997; Cole et al., J. CellBiol., 141:993-1008, 1998); many of these are now known to havehomologues in higher organisms, including humans. More recently, IFT hasbeen visualized in the ciliated sensory neurons of C. elegans (Orozco etal., Nature, 398:674, 1999; Signor et al., J. Cell Biol., 147:519-530,1999; Qin et al., Cur. Bio., 11: 1-20, 2001) and in the primary cilia ofmouse kidney cells (Pazour, manuscript in preparation) using GFP-taggedIFT proteins. Defects in IFT motors and particle proteins lead todefects in assembly of motile flagella in Chlamydomonas (Kozminski etal., J. Cell. Biol., 131:1517-1527, 1995; Pazour et al., J. Cell Biol.,144:473-481, 1999; Pazour et al., J. Cell Biol., 151:709-718, 2000), ofciliated sensory neurons in C. elegans (Perkins et al., Dev. Biol.,117:456-487, 1986; Cole et al., J. Cell Biol., 141:993-1008, 1998;Collet et al., Genetics, 148:187-200, 1998; Signor et al., J. CellBiol., 147:519-530, 1999; Wicks et al., Dev. Biol., 221:295-307, 2000;Qin et al., Cur. Bio., 11:1-20, 2001), and of nodal cilia (Nonaka etal., Cell, 95:829-837, 1998; Marszalek et al., Proc. Natl. Acad. Sci.U.S.A., 96:50435048, 1999; Takeda et al., J. Cell Biol., 145:825-836,1999; Murcia et al., Development, 127:2347-2355, 2000), kidney primarycilia (Pazour et al., J. Cell Biol., 151:709-718, 2000), and rod outersegments in mice (Pazour et al., manuscript in preparation). IFTfunctions in the transport of flagellar precursors to the tip of theflagellum, where they are needed for both flagellar assembly andmaintenance. In cilia and flagella with a sensory function, IFT can alsobe involved in signal transduction between the cilium or flagellum andthe cell body.

[0079] The Motors Powering IFT

[0080] The motors moving the IFT particles were defined by means ofChlamydomonas flagellar mutants defective in either kinesin-II orcytoplasmic dynein 1b.

[0081] The anterograde motor. Studies of flagellar assembly (fla)mutants in Chlamydomonas first identified the gene FLA10, which encodesa kinesin-II motor subunit (termed Fla10) present in the flagellum. IFTand Fla10 were essential for both the assembly and maintenance of theflagella. Inasmuch as kinesin-II moves towards the +ends of microtubules(Scholey, J. Cell Biol., 133:1-4, 1996), it was proposed thatanterograde IFT was powered by kinesin-II (Kozminski et al., J. CellBiol., 131:1517-1527, 1995). The flagellar Fla10-kinesin-II was purifiedto homogeneity and shown to be a typical heterotrimeric kinesin-11,composed of two motor subunits of 85 and 95 kD (also called Kif 3a and3b [Scholey, J. Cell Biol., 133:1-4, 1996]), and a non-motor subunit of115 kD, called KAP kinesin-associated protein, [Scholey, J. Cell Biol.,133:1-4, 1996]).

[0082] The Retrograde Motor.

[0083] Cytoplasmic dynein was first implicated in IFT when it was foundthat a mutation in Chlamydomonas LC8, a cytoplasmic dynein light chain,had short flagella that initially grew out to about one-half tothree-quarters normal length, and then gradually shortened (Pazour etal., 1998). These flagella exhibited normal levels of anterograde IFT,but had greatly reduced levels of retrograde IFT.

[0084] The Chlamydomonas dynein heavy chain isoform DHC1b forms theretrograde motor. In C. elegans, the homologue of DHC1b is Che-3, andmutations in Che-3 result in a phenotype very similar to that seen inDHC1b mutants in Chlamydomonas: the sensory cilia are very short andfilled with IFT particles (Collet et al., 1998; Wicks et al., 2000).Therefore, DHC1b (also known as DHC2 in mammals) is likely to be theretrograde IFT motor in all cilia and flagella.

[0085] IFT and the Tip of the Flagellum

[0086] IFT particles move all the way to the tip or base of theflagellum without pause; particles moving to the tip appear by DICmicroscopy to have more contrast than those moving to the base(Kozminski et al., 1993; Pipemo et al., 1998). FIG. 2 shows a model ofthis process. Analysis of Chlamydomonas IFT motor mutants indicates thatkinesin-II is carried as IFT cargo from the flagellar tip to the base(Pazour et al., 1998, 1999); conversely, because dyneins are minus-enddirected motors, it can be inferred that cytoplasmic dynein 1b iscarried as IFT cargo to the tip. There is evidence that flagellarprecursor proteins also are transported by IFT particles to the site ofaxonemal assembly at the flagellar tip, and released there (Pipemo etal., 1996). At the tip, the apparent size of the particles is reduced(possibly due to unloading of axonemal precursors), the kinesin motorbecomes cargo, and the cytoplasmic dynein 1b motor takes over totransport the particles back to the peri-basal body region.

[0087] The mechanisms by which IFT particle turnaround, cargo-release,and motor exchange occur at the tip are unknown, but by analogy withother bi-directional particle movement systems, e.g. melanophoremovement (Reese and Haimo, 2000), it may involve phosphorylation anddephosphorylation of motors and/or their associated proteins. Becausethe IFT particles move unidirectionally without stopping or reversing,the proteins that control the IFT motors must be highly localized at thetip and base of the flagellum. At the base of the flagellum, theregulatory proteins may be anchored to the transition fibers (Deane etal., manuscript in preparation). The tip of the flagellum also containsspecialized structures that may serve as anchors for the proteins thatturn kinesin off and dynein on: each of the A-subfibers of the outerdoublets is terminated by a plug which is connected to the flagellarmembrane by a thin filament (Dentler and Rosenbaum, 1977; Dentler,1989). The central pair of microtubules likewise is connected to the tipby a specific structure (Dentler and Rosenbaum, 1977), but this probablyis not involved in the turnaround process because Chlamydomonas mutantslacking the central pair have normal IFT (Kozminski et al., 1993).

[0088] Characterization of Chlamydomonas IFT particles

[0089] When fla10ts mutant cells are shifted to restrictive temperature,the number of IFT particles in the flagella decreases substantiallybefore the flagella are resorbed (Kozminski et al., 1995). By comparingthese fla10 mutant flagella to those of wild-type cells, it was possibleto determine which flagellar polypeptides make up the IFT particles. Inthis way, the IFT particles were found to sediment at 16 S in sucrosegradients and to be composed of at least 16 polypeptides (Piperno andMead, 1997; Cole et al., 1998), occurring in two complexes, termed A andB. Complex A contains 4 polypeptides of relatively high molecular weight(Mr 122-K144K), whereas Complex B contains 11-12 mostly lower MWpolypeptides (Mr <100K) (Cole et al. 1998). The identity of the proteinswas confirmed by analysis of LC8 mutant flagella, which accumulated boththe IFT polypeptides and IFT particles (Pazour et al., 1998).

[0090] Separation of the IFT particle polypeptides on two-dimensionalgels permitted microsequencing of individual proteins to obtain shortinternal amino-acid sequences (Cole et al., 1998). The peptide sequenceswere in turn used to generate PCR primers for amplification of cDNAsencoding several of the IFT particle polypeptides (see below). To date,the predicted sequences are novel. The sequences have permitted theidentification of Chlamydomonas insertional mutants lacking the genesencoding two of the IFT particle proteins, IFT57 and IFT88 (Pazour etal., 1999b; Pazour et al., 2000). These mutants grow and dividenormally, demonstrating that these IFT particle proteins are notinvolved in any essential processes. However, the IFT88 mutant fails toassemble flagella, whereas the IFT57 mutant assembles only very shortflagella. Thus, these specific IFT particle proteins are required forflagellar formation, although the difference in phenotypes suggests thatthe two proteins differ in their importance. In contrast to the DHC1bmutant, the IFT57 and IFT88 mutants do not accumulate IFT particles intheir flagella.

[0091] Localization of IFT Particle Proteins

[0092] Isolation of the IFT particles permitted the production of bothmonoclonal and polyclonal antibodies recognizing specific IFT particleproteins. Immunofluorescence microscopy using these antibodies and thoseprepared against the IFT motors revealed punctate staining along theflagella, presumably representing IFT particles in transit. However, theprincipal localization of both the IFT particle polypeptides and thekinesin and dynein motors was in a circular pattern around the two basalbodies/centrioles (Cole et al., 1998; Pazour et al., 1999). This wassomewhat surprising, because moving IFT particles had been observed onlyin the flagella by DIC microscopy, and the linear arrays of IFTparticles had been observed only in the flagella by electron microscopy.This notwithstanding, the immunoflourescence localization patternindicated that there is a large pool of IFT particle proteins and motorsat the base of the flagellum.

[0093] To learn more about this peri-basal body distribution of IFTparticle proteins, studies were carried out using gold-labeledantibodies and thin-sectioned material (Deane et al., manuscript inpreparation). These higher resolution studies revealed that the IFTproteins were localized at the “flagellar” end of the basal bodies,specifically on the membrane-associated ends of the transition fibersthat connect the basal body to the cell membrane (Ringo, 1967; Weiss etal., 1977). These fibers demarcate the boundary between the cytoplasmicand flagellar “compartments.” Although the flagellum is ostensibly“open” to the cytoplasm, it appears that only a subset of cytoplasmicproteins (the “flagellar” proteins) gain admission to the flagellarcompartment. Thus, the transition fibers may be structural components ofa filter or “flagellar pore” that controls movement of molecules andparticles between the cytoplasmic and flagellar compartments, much asthe nuclear pore controls movement between the cytoplasmic and nuclearcompartments. If access to the flagellar compartment is controlled, thenone would predict that pre-assembled flagellar structures, such asradial spokes (Diener et al., 1996) and dyneins arms (Fowkes andMitchell, 1998), either have flagellar localization signals on one ormore of their constituent polypeptides, or that they are escortedthrough the pore by a carrier perhaps the IFT particles—with which theyassociate. Therefore, the flagellar pore, where nonmembrane boundpolypeptides dock prior to gaining entrance to the flagellarcompartment, would be functionally similar to the nuclear pore.

[0094] A flagellar Pore Complex?

[0095] Immunofluorescence microscopy indicates that IFT particleproteins are localized primarily to the base of the Chlamydomonasflagellum, with only a few particles in the flagellum that mustrepresent IFT rafts in transit. Immuno-electron microscopy reveals thatthe IFT particle proteins are docked at the ends of “transition fibers”that extend from the distal end of the basal body to the plasma membraneat the base of the flagellum. These fibers, which connect each of thenine basal body triplet microtubules to the flagellar membrane,demarcate the boundary between the cytoplasmic and flagellarcompartments. The transition fibers thus may be the structural basis fora “flagellar pore complex” (FPC) that limits access to the flagellarcompartment (see FIG. 3). We propose that flagellar membrane andaxonemal proteins synthesized in the cytoplasm are transported to thebase of the flagellum, where they are recognized by IFT particleproteins and ushered through the FPC into the flagellar compartment.Transition fibers are present in association with all basal bodies, soit is expected that all cilia and flagella have an FPC that serves as agateway for admission of specific proteins to the cilium or flagellum.

[0096] IFT Particle Polypeptides in C. elegans

[0097] Database searching using the Chlamydomonas IFT particle proteinsequences revealed a large number of homologues in the nematode C.elegans for which there are known mutations. Interestingly, themutations are in the Che, Daf, and Osm genes that are required forformation and function of the worm sensory cilia. For example, Osm-1,Osm-5, and Osm-6 encode homologues of the Chlamydomonas IFT172, IFT88and IFT52 IFT particle proteins (Collet et al., 1998; Wicks et al.,2000; Qin et al., 2001). Therefore, IFT is essential for the assembly ofnon-motile sensory cilia in C. elegans. These findings, as well as thoseon Che-3, which encodes the retrograde IFT motor DHC 1b (see above),indicated that the process of IFT has been highly conserved throughoutevolution and is likely to be necessary for the assembly of all flagellaand cilia, including structures derived from these organelles.

[0098] It was presumed that, like the Chlamydomonas IFT particles, theIFT polypeptides initially localized in C. elegans sensory cilia moveanterogradely and retrogradely in the cilia. This has now been elegantlydemonstrated by fusing sequences encoding GFP to either the kinesin-IInon-motor subunit (KAP) gene or to IFT particle polypeptide genes,transforming the constructs into C. elegans, and observing the motilityof their products in vivo (Orozco et al., 1999; Signor et al., 1999).The rates of movement of IFT polypeptides and motors were similar toeach other, and were likewise similar to those of IFT particles inChlamydomonas flagella. Recently, several additional C. elegans IFTparticle polypeptides, contained in both IFT Complexes A and B, havebeen tagged with GFP and their motility observed in vivo. Complexes Aand B, and the motors that move them, were all shown to translocate atthe same rates in neuronal sensory cilia (Qin et al., 2001).

[0099] IFT and Polycystic Kidney Disease

[0100] Searching the databases with the sequences of the ChlamydomonasIFT particle proteins also revealed close homologues in higherorganisms, including mice and humans. The homologue of ChlamydomonasIFT88, termed Tg737 in mouse and man, was of particular interest,because an insertional mutation in this gene causes autosomal recessivepolycystic kidney disease (ARPKD) in the mouse (Moyer et al., 1994). Inboth the mouse and humans, ARPKD involves the formation of numerouscysts in the proximal and collecting tubules of the kidney (Grantham etal., 1996; Moyer et al., 1994). In humans, ARPKD affects up to 1 in10,000 newborns; most die within a few weeks of birth (Blythe andOckenden, 1971; Cole et al., 1987). ARPKD also may be responsible for ˜1in 3000 prenatal deaths and still births.

[0101] Although the kidney lacks motile cilia, many of the epithelialcells of the kidney collecting ducts and tubules have a single,non-motile ‘9+0’ cilium, called the primary cilium. Primary cilia are infact present on most cells in the body (seehttp://www.members.global2000.net/bowser/cilialist.html for acomprehensive list of cells having primary cilia), but in the kidneythey are particularly well developed. The fact that IFT88 is essentialfor flagellar formation in Chlamydomonas (see above) suggested that itsmammalian homologue, Tg737, might be important for formation of theprimary cilia in the kidney. Indeed, when kidneys of mice homozygous forthe Tg737 insertional mutation were examined by scanning electronmicroscopy, they were found to be defective in ciliary assembly (Pazouret al., 2000). Whereas wild-type mice had cilia extending severalmicrons into the lumens of the collecting ducts and tubules, the mutanthad only short stubs of cilia, just as in the case of the ChlamydomonasIFT88 deletion mutant. These results indicated that the primary cause ofARPKD in the mutant mouse is failure to assemble the primary cilium dueto a defect in an IFT particle protein. Subsequent studies have shownthat the Tg737 protein is concentrated at the base of the kidney cilia(Taulman et al., 2001), just as IFT particle proteins are located at thebase of the flagellum of Chlamydomonas, and that GFP-tagged Tg737 movesanterogradely and retrogradely in the cilia of wild-type kidney cells inculture (Pazour, unpublished results).

[0102] The function of the primary cilium is unknown (Alberts et al.,1994). In the kidney, the primary cilium may function as a flowmeter(Schwartz et al., 1997), as an osmometer, or as a chemodetector.Whatever its specific function, the results with the Tg737 mutant miceprovide the first evidence that it has a very important role in kidneyphysiology (Pazour et al., 2000).

[0103] A related renal disease, autosomal dominant polycystic kidneydisease (ADPKD), which affects up to 1 in 500 individuals, similarlyresults in the formation of renal cysts, although the symptoms may notbe clinically apparent until the patient reaches middle age (Grantham etal., 199_. The primary defects in the most common forms of ADPKD are ingenes coding for polycystin 1 (PKD1) and polycystin 2 (PKD2). PKD1 is anintegral membrane protein that directly interacts with PKD2 to form acalcium-sensitive cation channel probably acting in a sensory signalingpathway (Emmons and Somlo, 1999; Murcia et al., 1999; Somlo and Ehrlich,2001). How are the ADPKD polycystins and the defect in kidney ciliaassembly in ARPKD related? A clue to this was a report showing that theC. elegans homologues of the vertebrate polycystins are located on theworm's sensory cilia (Barr and Stemberg, 1999), where they also appearto be involved in signal transduction. More recent work indicates thatthe human polycystins likewise are displayed principally on the ciliarymembrane (Pazour et al., manuscript in preparation), suggesting that adefect in one of the polycystins impairs normal functioning of thekidney primary cilium and lead to ADPKD.

[0104] IFT Reveals a Role for Primary Cilia in Polycystic Kidney Disease

[0105] The cells of the proximal and distal collecting tubules of thevertebrate kidney each have a single nonmotile “9+0” primary ciliumextending from the apical cell surface into the lumen of the tubule. Thefunction of these primary cilia is unknown, but if their assembly isdisrupted in the mouse by a defect in a homologue of one of theChlamydomonas IFT particle polypeptides, autosomal recessive polycystickidney disease (ARPKD) results and the mice die during gestation or soonafter birth (Pazour et al., 2000). Therefore, vertebrate ARPKD occurswhen the kidney tubule primary cilia are missing or defective.

[0106] In humans the most common genetic deficiencies in a relateddisorder, autosomal dominant polycystic kidney disease (ADPKD), are inthe genes PKD 1 and PKD2, which encode polycystin 1 and 2, respectively(Somlo and Ehrlich, 2001). The two polycystins interact to form acalcium-activated cation channel involved in signal transduction(Gonzalez-Perrett et al., 2001).

[0107] The reason why either lack of the primary cilia or mutations inthe polycystins cause the polycystic kidney phenotype is likely to bebecause the polycystins on the cell surface are concentrated on themembranes of the kidney primary cilia (Pazour et al., manuscript inpreparation). This suggests that a defect in a polycystin impairsfunctioning of the primary cilium. Therefore, polycystic kidney diseasecan result either from mutations in the polycystins that are targeted tothe primary cilia, or from an inability to form the primary ciliathemselves.

[0108] IFT and Retinal Degenerative Disease

[0109] Primary cilia, or structures derived from primary cilia, also areinvolved in the development and function of several sensory structuresin the vertebrate body, e.g., in the retina, the inner ear, and thenasal epithelium. Inasmuch as IFT probably occurs in all cilia andflagella, it is likely to be important for the assembly and maintenanceof these sensory structures/tissues as well. The role of IFT has beenmost closely examined in retinal rod and cone outer segments. Theselight sensory dendritic processes, containing the photopigments andlight-transducing machinery, initially form from primary cilia (DeRobertis, 1956; Tokuyasu and Yamada, 1959); a short ‘9+0’ “connectingcilium” remains in the adult as the only path of communication betweenthe outer segment and the inner segment, where protein synthesis occurs(Young, 1976; Besharse, 1986). Following its formation, the rod outersegment turns over continuously at a high rate; it is estimated that2000 opsin molecules per minute are required to maintain the mammalianrod outer segment (Besharse, 1986), and all of this newly synthesizedprotein is likely to be transported to the outer segment through theconnecting cilium. A possible role for IFT in this process was firstindicated by the discovery that the IFT motor kinesin-II is present inthe connecting cilia of fish rods and cones (Beech et al., 1996).Subsequently, Cre-loxP mutagenesis was used to remove the kinesin-IImotor subunit, KIF3A, specifically from photoreceptor cells (Marszaleket al., 2000). In the absence of KIF3A, large quantities of opsin,arrestin and membranes accumulated in the inner segment, and thephotoreceptor cells eventually underwent apoptotic cell death. Theseresults implied that kinesin-II, present in the connecting cilium, waspowering IFT there and was required for the assembly and continuedmaintenance of the rod outer segment.

[0110] More recently, immunofluorescence microscopy has revealed thatseveral IFT particle proteins are concentrated at the proximal ends, andto a lesser extent the distal ends, of mouse rod connecting cilia(Pazour et al., 2000b), a distribution remarkably similar to that seenfor IFT particle proteins in Chlamydomonas. Moreover, in mice homozygousfor the insertional mutation in the IFT particle protein Tg737, the rodouter segments develop abnormally, and eventually degenerate, leading tocomplete disappearance of the rod cells (Pazour et al., manuscript inpreparation). These latter results provide very strong independentevidence that IFT occurs in the connecting cilium and has an importantrole in assembling and maintaining the rod outer segment, presumably bytransporting essential proteins from the inner segment to the outersegment (see FIG. 5). The degeneration of rod cells resulting fromdefects in IFT motors and particle proteins is very similar to thatobserved in retinitis pigmentosa and other human diseases causingprogressive blindness (Sung and Tai, 2000; Traboulsi, 1998). Therefore,the genes encoding IFT proteins must now be considered candidate genesfor these diseases.

[0111] IFT and Rod Outer Segments

[0112] Vertebrate rod outer segments are derived from ‘9+0’ primarycilia during embryogenesis of the retina. The distal portion of thecilium differentiates into folds that contain the visual pigments andthe phototransduction machinery. The proximal portion of the primarycilium remains as the “connecting cilium.” The connecting cilium is theonly connection between the rod inner segment, which contains all thecytoplasmic organelles, and the outer segment. In the adult, the outersegment turns over very rapidly due to shedding of membranous disks fromits distal tip. Both the initial development of the outer segment andits continued maintenance are dependent on the movement of precursorsfrom their site of synthesis in the inner segment, through theconnecting cilium to the outer segment.

[0113] Membrane proteins such as rhodopsin that are destined for theouter segment are synthesized in the endoplasmic reticulum, processedthrough the Golgi apparatus, and then transported in vesicles bycytoplasmic dynein 1 along microtubules that converge at the base of theconnecting cilium. There the vesicles fuse with the plasma membrane at aspecialized structure termed the periciliary ridge complex (Peters etal., 1983). Immunofluorescence microscopy has revealed a highconcentration of IFT particle proteins at the base of the connectingcilium, just as in Chlamydomonas (Pazour et al., 2000b). It ishypothesized that the rhodopsin and other outer segment precursorsbecome associated with IFT particles at the base of the connectingcilium, and are transported by kinesin II through the flagellar porecomplex and up the cilium to the base of the outer segment, where theyare released. The IFT particles are then transported by cytoplasmicdynein 2/1b back down the connecting cilium to the peri-basal bodyregion. Mutations in the carboxyl-terminal portion of rhodopsin causeabnormal transport and/or localization of rhodopsin, resulting inretinal degeneration (Sung and Tai, 2000; Tam et al., 2000). Similarly,mutations in kinesin-II or an IFT particle protein in the mouse resultin abnormal localization of rhodopsin and retinal degeneration(Marszalek et al., 2000; Pazour et al., manuscript in preparation).Thus, defects in IFT may be one cause of retinal degeneration in humans.

[0114] IFT is Required for Assembly of Nodal Cilia

[0115] As noted above, deletion of the IFT88 gene in Chlamydomonascompletely blocks flagellar assembly. Why, then, are connecting ciliaformed at all in rod cells of the mouse homozygous for an insertionalmutation in the IFT88 homologue, Tg737? The Tg737 insertional alleleused in the above studies results in expression of a smaller-than-normalTg737 protein, probably due to a defect in mRNA splicing (Taulman etal., 2001; G. Pazour, unpublished result). Apparently, this smallproduct is sufficient to support the assembly of the connecting cilium,but not adequate for full development and long-term maintenance of theouter segment. Interestingly, complete knockout of the mouse Tg737 geneis embryonic lethal (Murcia et al., 2000); the embryos lack nodal ciliaand are defective in left-right axis determination, a hallmark ofdefects in the nodal cilia (see “Defects in IFT reveal a role for nodalcilia in the development of left-right axis determination”). Therefore,Tg737 in the mouse, like its homologue IFT88 in Chlamydomonas, isessential for cilia formation. As in the case of the connecting cilium,the abnormal Tg737 protein in the original insertional mutant mouse mustbe sufficient to allow nodal cilia formation and normal embryogenesis.One also can infer that some cilia or structures derived fromcilia—viz., kidney primary cilia, rod outer segments—are more sensitiveto defects in IFT than other cilia—viz. nodal cilia and respiratorycilia (the latter are relatively unaffected in the original Tg737insertional mutant mouse.

[0116] Defects in IFT Reveal a Role for Embryonic Nodal Cilia inLeft-Right Axis Determination

[0117] Because IFT is required for the formation of all cilia andflagella, animals lacking IFT should be defective in all processes thatdepend on these organelles. Knockouts of IFT motors and particleproteins in the mouse reveal that one of the earliest roles for cilia inthe mammalian embryo is the development of left-right asymmetry. Thisprocess appears to depend on the motility of nodal cilia, which resembleprimary cilia but are unique in that they exhibit an unusual twirlingmovement not seen in other primary cilia. In the absence of the IFTmotor kinesin-II (Nonaka et al., 1998; Marzalek et al., 1999; Takeda etal., 1999) or the IFT particle protein Tg737 (Murcia et al., 2000),nodal cilia fail to develop. In these embryos, the earliest molecularmarkers of left-right asymmetry, which normally are expressed only inthe left lateral plate mesoderm shortly after development of the nodalcilia, are expressed bilaterally (Nonaka et al., 1998; Marszalek et al.,1999). Subsequently, the embryos undergo random laterality of heartlooping, so that in half of the embryos the heart is on the wrong sideof the midline, a condition known as situs inversus and frequentlyobserved in humans with defects in cilia function (Afzelius andMossberg, 1995). It has been proposed that the twirling movement of thenodal cilia sets up a gradient of a morphogen in the extraembryonicfluid across the node, resulting in left-right axis determination (Okadaet al., 1999; Supp et al., 1999). In support of such a role, embryos ofmice with a defect in left-right dynein, which may be the motor poweringnodal cilia movement, have non-motile nodal cilia and situs inversus(Okada et al., 1999; Supp et al., 1999). (Modified from Supp et al.,2000)

[0118] Further Examples of IFT Function

[0119] Intraflagellar Transport (IFT) is essential for both the initialassembly and subsequent maintenance of all motile (9+2) and sensory(9+0) cilia/flagella. The research shows that IFT is required for themovement of structural proteins, which form the microtubular axoneme, tothe flagellar tip, where assembly occurs. For those 9+2 cilia andflagella where motility is the principal function, e.g. flagella ofsperm, and cilia of the respiratory tract, oviduct, fimbriae, efferentducts of the testis, and ependymal cells lining the cavities of thespinal cord and brain, the motility function is dependent on properassembly and maintenance of the microtubule-containing axoneme. When thenodal cilia cannot form in the embryo, developmental diseases such assitus inversus result. IFT also is important for movement andmaintenance of proteins in the flagellar and ciliary membranes. Forexample, the membrane-associated proteins involved in phototransductionin the vertebrate retina are in the rod and cone outer segments, whichare modified cilia. Finally, IFT is important in the movement of signalsfrom the cilium to the cell body, as in the case of 9+0 primary ciliaand sensory cilia which monitor the enviromnent and relay thatinformation to the cell or nervous system.

[0120] II. DNA and Polypeptide Sequences

[0121] The present invention is based, at least in part, on thediscovery or characterization of a variety of cDNA and polypeptidemolecules, and sequences inferred from or homologous to them, whichencode proteins which are herein designated IFT 20, 27, 46, 52, 57, 72,88, 122, and 139, and Che-2. Sequences from Chlamydomonas,Caenorhabditis elegans, mouse, and human are included in the invention.These proteins exhibit a variety of physiological activities, and areincluded in a single application for the sake of convenience. It isunderstood that the allowability or nonallowability of claims directedto one of these proteins has no bearing on the allowability of claimsdirected to the others. The characteristics of each of these proteinsand the cDNAs encoding them are described separately in the ensuingsections. In addition to the full length mature and immature humanproteins described in the following sections, the invention includesfragments, derivatives, and variants of these proteins, as describedherein. These proteins, fragments, derivatives, and variants arecollectively referred to herein as polypeptides of the invention orproteins of the invention.

[0122] IFT27 is a member of the Rab family of small GTPases. Rab GTPasesand their effectors are generally involved in membrane vesicleformation, vesicle and organelle motility and transport, and tetheringof vesicles to their target compartment (Zerial and McBride, NatureReviews Molecular Cell Biology, 2:107-119, 2001). Known Rabs cyclebetween an “active” state in which GTP is bound, and an “inactive” statein which GDP is bound. Thus they serve as molecular “switches.” An“isolated nucleic acid molecule” is a nucleic acid molecule that isseparated from the 5′ and 3′ coding sequences with which it isimmediately contiguous in the naturally occurring genome of an organism.Isolated nucleic acid molecules include nucleic acid molecules that arenot naturally occurring, e.g., nucleic acid molecules created byrecombinant DNA techniques. Nucleic acid molecules include both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. Where single-stranded, the nucleic acid molecule maybe a sense strand or an antisense strand.

[0123] As used herein, a “signal sequence” includes a peptide of atleast about 15 or 20 amino acid residues in length which occurs at theN-terminus of secretory and membrane-bound proteins and which containsat least about 70% hydrophobic amino acid residues such as alanine,leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, orvaline. In a preferred embodiment, a signal sequence contains at leastabout 10 to 40 amino acid residues, preferably about 19-34 amino acidresidues, and has at least about 60-80%, more preferably at least about65-75%, and more preferably at least about 70% hydrophobic residues. Asignal sequence serves to direct a protein containing such a sequence toa lipid bilayer. A signal sequence is usually cleaved during processingof the mature protein.

[0124] The term “purified” as used herein refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.

[0125] Polypeptides or other compounds of interest are said to be“substantially pure” when they are within preparations that are at least60% by weight (dry weight) the compound of interest. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Purity canbe measured by any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0126] Where a particular polypeptide or nucleic acid molecule is saidto have a specific percent identity to a reference polypeptide ornucleic acid molecule of a defined length, the percent identity isrelative to the reference polypeptide or nucleic acid molecule. Thus, apeptide that is 50% identical to a reference polypeptide that is 100amino acids long can be a 50 amino acid polypeptide that is completelyidentical to a 50 amino acid long portion of the reference polypeptide.It might also be a 100 amino acid long polypeptide that is 50% identicalto the reference polypeptide over its entire length. Of course, manyother polypeptides will meet the same criteria. The same rule appliesfor nucleic acid molecules.

[0127] For polypeptides, the length of the reference polypeptidesequence will generally be at least 16 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids, 50 amino acids, or 100 amino acids. Fornucleic acids, the length of the reference nucleic acid sequence willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides or 300 nucleotides.

[0128] In the case of polypeptide sequences which are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0129] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). Preferably, the two sequences are the same length.

[0130] The determination of percent homology between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al. (1990) J Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to IFT 20, 27,46, 52, 57, 72, 88, 122, or 139, or Che-2 nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to IFT 20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2protein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search that detects distantrelationships between molecules. Id. When utilizing BLAST, Gapped BLAST,and PSI-Blast programs, the default parameters of the respectiveprograms (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0131] Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)). Such an algorithm is incorporated into the BestFitprogram, which is part of the Wisconsin™ package, and is used to findthe best segment of similarity between two sequences. BestFit reads ascoring matrix that contains values for every possible GCG symbol match.The program uses these values to construct a path matrix that representsthe entire surface of comparison with a score at every position for thebest possible alignment to that point. The quality score for the bestalignment to any point is equal to the sum of the scoring matrix valuesof the matches in that alignment, less the gap creation penaltymultiplied by the number of gaps in that alignment, less the gapextension penalty multiplied by the total length of all gaps in thatalignment. The gap creation and gap extension penalties are set by theuser. If the best path to any point has a negative value, a zero is putin that position.

[0132] After the path matrix is complete, the highest value on thesurface of comparison represents the end of the best region ofsimilarity between the sequences. The best path from this highest valuebackwards to the point where the values revert to zero is the alignmentshown by BestFit. This alignment is the best segment of similaritybetween the two sequences. Further documentation can be found athttp://ir.ucdavis.edulGCGhelp/bestfit.html#algorithm.

[0133] Additional algorithms for sequence analysis are known in the artand include ADVANCE and ADAM as described in Torellis and Robotti (1994)Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a controloption that sets the sensitivity and speed of the search. If ktup=2,similar regions in the two sequences being compared are found by lookingat pairs of aligned residues; if ktup=1, single aligned amino acids areexamined. ktup can be set to 2 or 1 for protein sequences, or from 1 to6 for DNA sequences. The default if ktup is not specified is 2 forproteins and 6 for DNA. For a further description of FASTA parameters,see http://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, thecontents of which are incorporated herein by reference.

[0134] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

[0135] As used herein, the phrase “allelic variant” refers to anucleotide sequence that occurs at a given locus or to a polypeptideencoded by the nucleotide sequence. Allelic variants of any of thesegenes can be identified by sequencing the corresponding chromosomalportion at the indication location in multiple individuals.

[0136] Nucleic Acids Encoding IFT Particle Proteins

[0137] The invention encompasses nucleic acids that have a sequence thatis substantially identical to the nucleic acid sequence of ChlamydomonasIFT particle protein genes 20, 27, 46, 52, 57, 72, 88, 122, or 139, orChe-2, as well as homologous mouse and human sequences. A nucleic acidsequence which is substantially identical to a given reference nucleicacid sequence is hereby defined as a nucleic acid having a sequence thathas at least 85%, preferably 90%, and more preferably 95%, 98%, 99% ormore identity to the sequence of the given reference nucleic acidsequence.

[0138] The IFT 20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2 nucleicacid molecules of the invention can be cDNA, genomic DNA, synthetic DNA,or RNA, and can be double-stranded or single-stranded (i. e., either asense or an antisense strand). Fragments of these molecules are alsoconsidered within the scope of the invention, and can be produced, forexample, by the polymerase chain reaction (PCR) or generated bytreatment with one or more restriction endonucleases. A ribonucleic acid(RNA) molecule can be produced by in vitro transcription.

[0139] The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. In addition, these nucleic acid molecules are notlimited to sequences that only encode polypeptides, and thus, caninclude some or all of the non-coding sequences that lie upstream ordownstream from a coding sequence.

[0140] The nucleic acid molecules of the invention can be synthesized(for example, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, mouse, rat, guinea pig, cow, sheep, horse, pig,rabbit, monkey, dog, or cat. Combinations or modifications of thenucleotides within these types of nucleic acids are also encompassed.

[0141] In addition, the isolated nucleic acid molecules of the inventionencompass fragments that are not found as such in the natural state.Thus, the invention encompasses recombinant molecules, such as those inwhich a nucleic acid molecule (for example, an isolated nucleic acidmolecule encoding IFT 20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2)is incorporated into a vector (for example, a plasmid or viral vector)or into the genome of a heterologous cell (or the genome of a homologouscell, at a position other than the natural chromosomal location).Recombinant nucleic acid molecules and uses therefor are discussedfurther below.

[0142] In the event the nucleic acid molecules of the invention encodeor act as antisense molecules, they can be used for example, to regulatetranslation of mRNA of the invention. Techniques associated withdetection or regulation of expression of nucleic acids or polypeptidesof the invention are well known to skilled artisans and can be used todiagnose and/or treat disorders associated with aberrant expression ofnucleic acids or polypeptides of the invention.

[0143] The invention also encompasses nucleic acid molecules thathybridize under stringent conditions to a nucleic acid molecule encodinga polypeptide of the invention. The cDNA sequences described herein canbe used to identify these hybridizing nucleic acids, which include, forexample, nucleic acids that encode homologous polypeptides in otherspecies, and splice variants of the genes of the invention in humans orother mammals. Accordingly, the invention features methods of detectingand isolating these nucleic acid molecules. Using these methods, asample (for example, a nucleic acid library, such as a cDNA or genomiclibrary) is contacted (or “screened”) with a probe specific to anucleotide of the invention. The probe will selectively hybridize tonucleic acids encoding related polypeptides (or to complementarysequences thereof). The probe, which can contain at least 25 (forexample, 25, 50, 100, or 200 nucleotides) can be produced using any ofseveral standard methods (see, for example, Ausubel et al., “CurrentProtocols in Molecular Biology, Vol. I,” Green Publishing Associates,Inc., and John Wiley & Sons, Inc., NY, 1989). For example, the probe canbe generated using PCR amplification methods in which oligonucleotideprimers are used to amplify a nucleic acid sequence specific to anucleic acid of the invention that can be used as a probe to screen anucleic acid library and thereby detect nucleic acid molecules (withinthe library) that hybridize to the probe.

[0144] One single-stranded nucleic acid is said to hybridize to anotherif a duplex forms between them. This occurs when one nucleic acidcontains a sequence that is the reverse and complement of the other(this same arrangement gives rise to the natural interaction between thesense and antisense strands of DNA in the genome and underlies theconfiguration of the “double helix”). Complete complementarity betweenthe hybridizing regions is not required in order for a duplex to form;it is only necessary that the number of paired bases is sufficient tomaintain the duplex under the hybridization conditions used.

[0145] Typically, hybridization conditions are of low to moderatestringency. These conditions favor specific interactions betweencompletely complementary sequences, but allow some non-specificinteraction between less than perfectly matched sequences to occur aswell. After hybridization, the nucleic acids can be “washed” undermoderate or high conditions of stringency to dissociate duplexes thatare bound together by some non-specific interaction (the nucleic acidsthat form these duplexes are thus not completely complementary).

[0146] As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (for example,room temperature) using a solution containing a salt concentration thatis equivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

[0147] In reactions where nucleic acids are hybridized, the conditionsused to achieve a given level of stringency will vary. There is not oneset of conditions, for example, that will allow duplexes to form betweenall nucleic acids that are 85% identical to one another; hybridizationalso depends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (for example, the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (for example, DNA or RNA)affect hybridization. An additional consideration is whether one of thenucleic acids is immobilized (for example, on a filter).

[0148] An example of a progression from lower to higher stringencyconditions is the following, where the salt content is given as therelative abundance of SSC (a salt solution containing sodium chlorideand sodium citrate; 2× SSC is 10-fold more concentrated than 0.2× SSC).Nucleic acids are hybridized at 42° C. in 2× SSC/0.1% SDS (sodiumdodecylsulfate; a detergent) and then washed in 0.2× SSC/0.1% SDS atroom temperature (for conditions of low stringency); 0.2× SSC/0.1% SDSat 42° C. (for conditions of moderate stringency); and 0.1× SSC at 68°C. (for conditions of high stringency). Washing can be carried out usingonly one of the conditions given, or each of the conditions can be used(for example, washing for 10- 15 minutes each in the order listedabove). Any or all of the washes can be repeated. As mentioned above,optimal conditions will vary and can be determined empirically.

[0149] A second set of conditions that are considered “stringentconditions” are those in which hybridization is carried out at 50° C. inChurch buffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% BSA) and washing iscarried out at 50° C. in 2× SSC.

[0150] Once detected, the nucleic acid molecules can be isolated by anyof a number of standard techniques (see, for example, Sambrook et al.,“Molecular Cloning, A Laboratory Manual,” 2nd Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

[0151] The invention also encompasses: (a) expression vectors thatcontain any of the foregoing coding sequences (related to a polypeptideof the invention) and/or their complements (that is, “antisense”sequence); (b) expression vectors that contain any of the foregoingcoding sequences (related to a polypeptide of the invention) operativelyassociated with a regulatory element (examples of which are given below)that directs the expression of the coding sequences; (c) expressionvectors containing, in addition to sequences encoding a polypeptide ofthe invention, nucleic acid sequences that are unrelated to nucleic acidsequences encoding a polypeptide of the invention, such as moleculesencoding a reporter or marker; and (d) genetically engineered host cellsthat contain any of the foregoing expression vectors and thereby expressthe nucleic acid molecules of the invention in the host cell.

[0152] Recombinant nucleic acid molecules can contain a sequenceencoding a soluble polypeptide of the invention; mature polypeptide ofthe invention; or polypeptide of the invention having an added orendogenous signal sequence. A full length polypeptide of the invention;a domain of a polypeptide of the invention; or a fragment thereof may befused to additional polypeptides, as described below. Similarly, thenucleic acid molecules of the invention can encode the mature form of apolypeptide of the invention or a form that encodes a polypeptide thatfacilitates secretion. In the latter instance, the polypeptide istypically referred to as a proprotein, which can be converted into anactive form by removal of the signal sequence, for example, within thehost cell. Proproteins can be converted into the active form of theprotein by removal of the inactivating sequence.

[0153] The regulatory elements referred to above include, but are notlimited to, inducible and non-inducible promoters, enhancers, operatorsand other elements, which are known to those skilled in the art, andwhich drive or otherwise regulate gene expression. Such regulatoryelements include but are not limited to the cytomegalovirus hCMVimmediate early gene, the early or late promoters of SV40 adenovirus,the lac system, the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage A, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase, thepromoters of acid phosphatase, and the promoters of the yeast α-matingfactors.

[0154] Similarly, the nucleic acid can form part of a hybrid geneencoding additional polypeptide sequences, for example, sequences thatfunction as a marker or reporter. Examples of marker or reporter genesinclude β-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a polypeptide of theinvention and the second portion being, for example, the reporterdescribed above or an immunoglobulin constant region.

[0155] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (for example, E. coli and B. subtilis) transformed withrecombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expressionvectors containing the nucleic acid molecules of the invention; yeast(for example, Saccharomyces and Pichia) transformed with recombinantyeast expression vectors containing the nucleic acid molecules of theinvention (preferably containing the nucleic acid sequence encoding apolypeptide of the invention); insect cell systems infected withrecombinant virus expression vectors (for example, baculovirus)containing the nucleic acid molecules of the invention; plant cellsystems infected with recombinant virus expression vectors (for example,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) ortransformed with recombinant plasmid expression vectors (for example, Tiplasmid) containing nucleotide sequences of nucleic acids of theinvention; or mammalian cell systems (for example, COS, CHO, BHK, 293,VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (for example, the metallothionein promoter) or frommammalian viruses (for example, the adenovirus late promoter and thevaccinia virus 7.5K promoter).

[0156] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions containing polypeptides of the invention or for raisingantibodies to those polypeptides, vectors that are capable of directingthe expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J.2:1791, 1983), in which the coding sequence of the insert may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye,Nucleic Acids Res. 13:3101-3109, 1985; Van Heeke and Schuster, J. Biol.Chem. 264:5503-5509, 1989); and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0157] In an insect system, Autographa californica nuclear polyhidrosisvirus (AcNPV) can be used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The coding sequence of theinsert may be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of the coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (for example, see Smithet al., J. Virol. 46:584, 1983; Smith, U.S. Patent No. 4,215,051).

[0158] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the nucleic acid molecule of the invention may beligated to an adenovirus transcription/translation control complex, forexample, the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (for example, region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing a gene product of the inventionin infected hosts (for example, see Logan and Shenk, Proc. Natl. Acad.Sci. USA 81:3655-3659, 1984). Specific initiation signals may also berequired for efficient translation of inserted nucleic acid molecules.These signals include the ATG initiation codon and adjacent sequences.In cases where an entire gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of the coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:516-544, 1987).

[0159] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (forexample, glycosylation) and processing (for example, cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Themammalian cell types listed above are among those that can serve assuitable host cells.

[0160] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the sequences of nucleic acids or polypeptides of the inventiondescribed above may be engineered. Rather than using expression vectorsthat contain viral origins of replication, host cells can be transformedwith DNA controlled by appropriate expression control elements (forexample, promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. This method can advantageously beused to engineer cell lines that express nucleic acids or polypeptidesof the invention. Such engineered cell lines may be particularly usefulin screening and evaluation of compounds that affect the endogenousactivity of the gene product.

[0161] A number of selection systems can be used. For example, theherpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA78:2072, 1981); neo, which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147,1984).

[0162] The nucleic acid molecules of the invention are useful fordiagnosis of disorders associated with aberrant expression of nucleicacid molecules of the invention are also useful in genetic mapping andchromosome identification.

[0163] IFT Particle Polypeptides

[0164] The invention also includes polypeptides that have a sequencethat is substantially identical to the amino acid sequence ofChlamydomonas IFT particle polypeptides 20, 27, 46, 52, 57, 72, 88, 122,or 139, or Che-2. A polypeptide which is “substantially identical” to agiven reference polypeptide is a polypeptide having a sequence that hasat least 85%, preferably 90%, and more preferably 95%, 98%, 99% or moreidentity to the sequence of the given reference polypeptide sequence.

[0165] The terms “protein” and “polypeptide” are used hereininterchangably to describe any chain of amino acids, regardless oflength or post-translational modification (for example, glycosylation orphosphorylation). Thus, the term “IFT 20, 27, 46, 52, 57, 72, 88, 122,or 139, or Che-2 polypeptide” includes: full-length, naturally occurringprotein of the invention; recombinantly or synthetically producedpolypeptide that corresponds to a full-length naturally occurringprotein of the invention; or particular domains or portions of thenaturally occurring protein. The term also encompasses mature apolypeptide of the invention that has an added amino-terminal methionine(useful for expression in prokaryotic cells).

[0166] The polypeptides of the invention described herein are thoseencoded by any of the nucleic acid molecules described above and includefragments, mutants, truncated forms, and fusion proteins of polypeptidesof the invention. These polypeptides can be prepared for a variety ofuses, including but not limited to the generation of antibodies, asreagents in diagnostic assays, for the identification of other cellulargene products or compounds that can modulate the activity or expressionof nucleic acids or polypeptides of the invention, and as pharmaceuticalreagents useful for the treatment of disorders associated with aberrantexpression or activity of nucleic acids or polypeptides of theinvention.

[0167] Preferred polypeptides are substantially pure polypeptides of theinvention, including those that correspond to the polypeptide with anintact signal sequence, and the secreted form of the polypeptide.Especially preferred are polypeptides that are soluble under normalphysiological conditions.

[0168] The invention also encompasses polypeptides that are functionallyequivalent to polypeptides of the invention. These polypeptides areequivalent to polypeptides of the invention in that they are capable ofcarrying out one or more of the functions of polypeptides of theinvention in a biological system. Preferred polypeptides of theinvention have 20%, 40%, 50%, 75%, 80%, or even 90% of one or more ofthe biological activities of the full-length, mature human form ofpolypeptides of the invention. Such comparisons are generally based onan assay of biological activity in which equal concentrations of thepolypeptides are used and compared. The comparison can also be based onthe amount of the polypeptide required to reach 50% of the maximalstimulation obtainable.

[0169] Functionally equivalent proteins can be those, for example, thatcontain additional or substituted amino acid residues. Substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0170] Polypeptides that are functionally equivalent to polypeptides ofthe invention can be made using random mutagenesis techniques well knownto those skilled in the art. It is more likely, however, that suchpolypeptides will be generated by site-directed mutagenesis (again usingtechniques well known to those skilled in the art). These polypeptidesmay have increased functionality or decreased functionality.

[0171] To design functionally equivalent polypeptides, it is useful todistinguish between conserved positions and variable positions. This canbe done by aligning the amino acid sequence of a protein of theinvention from one species with its homolog from another species.Skilled artisans will recognize that conserved amino acid residues aremore likely to be necessary for preservation of function. Thus, it ispreferable that conserved residues are not altered.

[0172] Mutations within the coding sequence of nucleic acid molecules ofthe invention can be made to generate variant genes that are bettersuited for expression in a selected host cell. For example, N-linkedglycosylation sites can be altered or eliminated to achieve, forexample, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionsof any one or more of the glycosylation recognition sequences whichoccur, and/or an amino acid deletion at the second position of any oneor more of such recognition sequences, will prevent glycosylation at themodified tripeptide sequence (see, for example, Miyajima et al., EMBOJ.5:1193, 1986).

[0173] The polypeptides of the invention can be expressed fused toanother polypeptide, for example, a marker polypeptide or fusionpartner. For example, the polypeptide can be fused to a hexa-histidinetag to facilitate purification of bacterially expressed protein or ahemagglutinin tag to facilitate purification of protein expressed ineukaryotic cells.

[0174] A fusion protein may be readily purified by utilizing an antibodyspecific for the fusion protein being expressed. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Proc. Natl.Acad. Sci. USA 88: 8972-8976, 1991). In this system, the gene ofinterest is subcloned into a vaccinia recombination plasmid such thatthe gene's open reading frame is translationally fused to anaminoterminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

[0175] The polypeptides of the invention can be chemically synthesized(for example, see Creighton, “Proteins: Structures and MolecularPrinciples,” W.H. Freeman & Co., NY, 1983), or, perhaps moreadvantageously, produced by recombinant DNA technology as describedherein. For additional guidance, skilled artisans may consult Ausubel etal. (supra), Sambrook et al. (“Molecular Cloning, A Laboratory Manual,”Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), and,particularly for examples of chemical synthesis Gait, M. J. Ed.(“Oligonucleotide Synthesis,” IRL Press, Oxford, 1984).

[0176] The invention also features polypeptides that interact withnucleic acids or polypeptides of the invention (and the genes thatencode them) and thereby alter the function of nucleic acids orpolypeptides of the invention. Interacting polypeptides can beidentified using methods known to those skilled in the art. One suitablemethod is the “two-hybrid system,” which detects protein interactions invivo (Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578, 1991). A kitfor practicing this method is available from Clontech (Palo Alto,Calif.).

[0177] Transgenic Animals

[0178] Polypeptides of the invention can also be expressed in transgenicanimals. These animals represent a model system for the study ofdisorders that are caused by or exacerbated by overexpression orunderexpression of nucleic acids or polypeptides of the invention, andfor the development of therapeutic agents that modulate the expressionor activity of nucleic acids or polypeptides of the invention.

[0179] Transgenic animals can be farm animals (pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (for example, baboons, monkeys, andchimpanzees), and domestic animals (for example, dogs and cats).Transgenic mice are especially preferred.

[0180] Any technique known in the art can be used to introduce an IFT20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2 transgene into animalsto produce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (U.S. Pat.No. 4,873,191); retrovirus mediated gene transfer into germ lines (Vander Putten et al., Proc. Natl. Acad. Sci., USA 82:6148, 1985); genetargeting into embryonic stem cells (Thompson et al., Cell 56:313,1989); and electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803,1983).

[0181] The present invention provides for transgenic animals that carrya transgene of the invention in all their cells, as well as animals thatcarry a transgene in some, but not all of their cells. That is, theinvention provides for mosaic animals. The transgene can be integratedas a single transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene can also be selectively introducedinto and activated in a particular cell type (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232, 1992). The regulatory sequences required forsuch a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart.

[0182] When it is desired that the transgene of the invention beintegrated into the chromosomal site of the endogenous gene, genetargeting is preferred. Briefly, when such a technique is to be used,vectors containing some nucleotide sequences homologous to an endogenousgene of the invention are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous gene. Thetransgene also can be selectively introduced into a particular celltype, thus inactivating the endogenous gene of the invention in onlythat cell type (Gu et al., Science 265:103, 1984). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art. These techniques are useful for preparing“knock outs” lacking a functional gene.

[0183] Once transgenic animals have been generated, the expression ofthe recombinant gene of the invention can be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to determine whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Biological samples can also be evaluatedimmunocytochemically using antibodies specific for the product of thetransgene of the invention. Samples of tissue expressing the gene of theinvention can also be evaluated immunocytochemically using antibodiesspecific for the product of the transgene of the invention.

[0184] For a review of techniques that can be used to generate andassess transgenic animals, skilled artisans can consult Gordon (Intl.Rev. Cytol. 115:171-229, 1989), and may obtain additional guidance from,for example: Hogan et al. “Manipulating the Mouse Embryo” (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1986; Krimpenfort et al.,Bio/Technology 9:86, 1991; Palmiter et al., Cell 41:343, 1985; Kraemeret al., “Genetic Manipulation of the Early Mammalian Embryo,” ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1985; Hammer et al.,Nature 315:680, 1985; Purcel et al., Science, 244:1281, 1986; Wagner etal., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.5,175,384 (the latter two publications are hereby incorporated byreference).

[0185] Anti-IFT 20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2Antibodies

[0186] Human polypeptides of the invention (or immunogenic fragments oranalogs) can be used to raise antibodies useful in the invention; suchpolypeptides can be produced by recombinant techniques or synthesized(see, for example, “Solid Phase Peptide Synthesis,” supra; Ausubel etal., supra). In general, the peptides can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Antibodies can be purifiedby peptide antigen affinity chromatography.

[0187] In particular, various host animals can be immunized by injectionwith a polypeptide of the invention. Host animals include rabbits, mice,guinea pigs, and rats. Various adjuvants that can be used to increasethe immunological response depend on the host species and includeFreund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Potentially useful human adjuvantsinclude BCG (bacille Calmette-Guerin) and Corynebacterium parvum.Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.

[0188] Antibodies within the invention therefore include polyclonalantibodies and, in addition, monoclonal antibodies, humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, and molecules produced using a Fab expression library.

[0189] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using thepolypeptides of the invention described above and standard hybridomatechnology (see, for example, Kohler et al., Nature 256:495, 1975;Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J.Immunol. 6:292, 1976; Hammerling et al., “Monoclonal Antibodies and TCell Hybridomas,” Elsevier, N.Y., 1981; Ausubel et al., supra).

[0190] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.,Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole etal., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this a particularly useful method of production.

[0191] Once produced, polyclonal or monoclonal antibodies are tested forspecific recognition of polypeptides of the invention by Western blot orimmunoprecipitation analysis by standard methods, e.g., as described inAusubel et al., supra. Antibodies that specifically recognize and bindto polypeptides of the invention are useful in the invention. Forexample, such antibodies can be used in an immunoassay to monitor thelevel of a polypeptide of the invention produced by a mammal (forexample, to determine the amount or subcellular location of apolypeptide of the invention).

[0192] Preferably, antibodies of the invention are produced usingfragments of the protein of the invention that lie outside highlyconserved regions and appear likely to be antigenic, by criteria such ashigh frequency of charged residues. In one specific example, suchfragments are generated by standard techniques of PCR, and are thencloned into the pGEX expression vector (Ausubel et al., supra). Fusionproteins are expressed in E. coli and purified using a glutathioneagarose affinity matrix as described in Ausubel, et al., supra.

[0193] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusions can be generated for each protein,and each fusion can be injected into at least two rabbits. Antisera canbe raised by injections in a series, preferably including at least threebooster injections.

[0194] Antisera may also checked for its ability to immunoprecipitaterecombinant proteins of the invention or control proteins, such asglucocorticoid receptor, CAT, or luciferase.

[0195] The antibodies can be used, for example, in the detection of thepolypeptide of the invention in a biological sample as part of adiagnostic assay. Antibodies also can be used in a screening assay tomeasure the effect of a candidate compound on expression or localizationof a polypeptide of the invention. Additionally, such antibodies can beused in conjunction with the gene therapy techniques described to, forexample, evaluate normal and/or genetically engineered cells thatexpress nucleic acids or polypeptides of the invention prior to theirintroduction into the patient. Such antibodies additionally can be usedin a method for inhibiting abnormal activity of nucleic acids orpolypeptides of the invention.

[0196] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.

[0197] Generally, partially human antibodies and fully human antibodieshave a longer half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration are oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

[0198] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692)can be adapted to produce single chain antibodies against polypeptidesof the invention. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

[0199] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0200] Antibodies to polypeptides of the invention can, in turn, be usedto generate anti-idiotype antibodies that resemble a portion of theprotein of the invention using techniques well known to those skilled inthe art (see, e.g., Greenspan et al., FASEB J 7:437, 1993; Nissinoff, J.Immunol. 147:2429, 1991). For example, antibodies that bind to theprotein of the invention and competitively inhibit the binding of abinding partner of the protein can be used to generate anti-idiotypesthat resemble a binding partner binding domain of the protein and,therefore, bind and neutralize a binding partner of the protein. Suchneutralizing anti-idiotypic antibodies or Fab fragments of suchanti-idiotypic antibodies can be used in therapeutic regimens.

[0201] Antibodies can be humanized by methods known in the art. Forexample, monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and 5,569,825,both of which are hereby incorporated by reference).

[0202] The methods described herein in whichanti-polypeptide-of-the-invention antibodies are employed may beperformed, for example, by utilizing pre-packaged diagnostic kitscomprising at least one specific polypeptide-of-the-invention antibodyreagent described herein, which may be conveniently used, for example,in clinical settings, to diagnose patients exhibiting symptoms ofdisorders associated with aberrant expression of nucleic acids orpolypeptides of the invention.

[0203] An antibody (or fragment thereof) can be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive agent (e.g., a radioactive metal ion). Cytotoxins andcytotoxic agents include any agent that is detrimental to cells.Examples of such agents include taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6thioguanine, cytarabine, and5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin {formerly designated daunomycin} anddoxorubicin), antibiotics (e.g., dactinomycin {formerly designatedactinomycin}, bleomycin, mithramycin, and anthramycin), and anti-mitoticagents (e.g., vincristine and vinblastine).

[0204] Conjugated antibodies of the invention can be used for modifyinga given biological response, the drug moiety not being limited toclassical chemical therapeutic agents. For example, the drug moiety canbe a protein or polypeptide possessing a desired biological activity.Such proteins include, for example, toxins such as abrin, ricin A,Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumornecrosis factor, alpha-interferon, beta-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; andbiological response modifiers such as lymphokines, interleukin-1,interleukin-2, interleukin-6, granulocyte macrophage colony stimulatingfactor, granulocyte colony stimulating factor, or other growth factors.

[0205] Techniques for conjugating a therapeutic moiety to an antibodyare well known (see, e.g., Arnon et al., 1985, “Monoclonal AntibodiesFor Immunotargeting Of Drugs In Cancer Therapy”, in MonoclonalAntibodies And Cancer Therapy, Reisfeld et al., Eds., Alan R. Liss, Inc.pp. 243-256; Hellstrom et al., 1987, “Antibodies For Drug Delivery”, inControlled Drug Delivery, 2nd ed., Robinson et al., Eds., Marcel Dekker,Inc., pp. 623-653; Thorpe, 1985, “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review”, in Monoclonal Antibodies ′84: BiologicalAnd Clinical Applications, Pinchera et al., Eds., pp. 475-506;“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., Eds., Academic Press, pp.303-316, 1985; and Thorpe et al., 1982, Immunol. Rev., 62:119-158).Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

[0206] Antisense Nucleic Acids

[0207] Treatment regimes based on an “antisense” approach involve thedesign of oligonucleotides (either DNA or RNA) that are complementary tomRNA of the invention. These oligonucleotides bind to the complementarymRNA transcripts of the invention and prevent translation. Absolutecomplementarity, although preferred, is not required. A sequence“complementary” to a portion of an RNA, as referred to herein, means asequence having sufficient complementarily to be able to hybridize withthe RNA, forming a stable duplex; in the case of double-strandedantisense nucleic acids, a single strand of the duplex DNA may betested, or triplex formation may be assayed. The ability to hybridizewill depend on both the degree of complementarily and the length of theantisense nucleic acid. Generally, the longer the hybridizing nucleicacid, the more base mismatches with an RNA it may contain and still forma stable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0208] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs recently have been shown to be effective atinhibiting translation of mRNAs as well (Wagner, Nature 372:333, 1984).Thus, oligonucleotides complementary to either the 5′ or 3′nontranslated, non-coding regions of the gene or mRNA can be used in anantisense approach to inhibit translation of endogenous mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon.

[0209] Antisense oligonucleotides complementary to mRNA coding regionsare less efficient inhibitors of translation but can be used inaccordance with the invention. Whether designed to hybridize to the 5′,3′, or coding region of an mRNA, antisense nucleic acids should be atleast six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides, or at least 50 nucleotides.

[0210] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

[0211] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (as described, e.g., in Letsinger et al., Proc. Natl. Acad.Sci. USA 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brainbarrier (see, for example, PCT Publication No. WO 89/10134), orhybridization-triggered cleavage agents (see, for example, Krol et al.,BioTechniques 6:958, 1988), or intercalating agents (see, for example,Zon, Pharm. Res. 5:539, 1988). To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

[0212] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

[0213] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0214] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal, or ananalog of any of these backbones.

[0215] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids. Res. 15:6625, 1987). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett. 215:327,1987).

[0216] Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). As examples, phosphorothioate oligonucleotides can besynthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209,1988), and methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., Proc. Natl. AcadSci. USA 85:7448, 1988).

[0217] The antisense molecules should be delivered to cells that expressnucleic acids or polypeptides of the invention in vivo. A number ofmethods have been developed for delivering antisense DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systemically.

[0218] However, it is often difficult to achieve intracellularconcentrations of the antisense molecule sufficient to suppresstranslation of endogenous mRNAs. Therefore, a preferred approach uses arecombinant DNA construct in which the antisense oligonucleotide isplaced under the control of a strong pol III or pol II promoter. The useof such a construct to transfect target cells in the patient will resultin the transcription of sufficient amounts of single stranded RNAs thatwill form complementary base pairs with the endogenous transcripts ofnucleic acids of the invention and thereby prevent translation of theendogenous mRNA. For example, a vector can be introduced in vivo suchthat it is taken up by a cell and directs the transcription of anantisense RNA. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA.

[0219] Such vectors can be constructed by recombinant DNA technologymethods standard in the art. Vectors can be plasmid, viral, or othersknown in the art, used for replication and expression in mammaliancells. Expression of the sequence encoding the antisense RNA can be byany promoter known in the art to act in mammalian, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to: the SV40 early promoter region (Bemoistet al., Nature 290:304, 1981); the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797,1988); the herpes thymidine kinase promoter (Wagner et al., Proc. Natl.Acad. Sci. USA 78:1441, 1981); or the regulatory sequences of themetallothionein gene (Brinster et al., Nature 296:39, 1988).

[0220] Ribozymes

[0221] Ribozyme molecules designed to catalytically cleave mRNAtranscripts of nucleic acids of the invention can be used to preventtranslation and expression of mRNA of the invention. (see, e.g., PCTPublication WO 90/11364; Saraver et al., Science 247:1222, 1990). Whilevarious ribozymes that cleave mRNA at site-specific recognitionsequences can be used to destroy mRNAs of the invention, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art (Haseloff etal., Nature 334:585, 1988). There are numerous examples of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofhuman cDNA of the invention. Preferably, the ribozyme is engineered sothat the cleavage recognition site is located near the 5′ end of themRNA, i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[0222] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena thermophila (known as the IVS orL-19 IVS RNA), and which has been extensively described by Cech and hiscollaborators (Zaug et al., Science 224:574, 1984; Zaug et al., Science,231:470, 1986; Zug et al., Nature 324:429, 1986; PCT Application No. WO88/04300; and Been et al., Cell 47:207, 1986). The Cech-type ribozymeshave an eight base-pair sequence that hybridizes to a target RNAsequence, whereafter cleavage of the target RNA takes place. Theinvention encompasses those Cech-type ribozymes that target eightbase-pair active site sequences present in nucleic acids of theinvention.

[0223] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.), and should be delivered to cells which express nucleic acids orpolypeptides of the invention in vivo. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous messages and inhibit translation. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

[0224] Other Methods for Modulating IFT 20, 27, 46, 52, 57, 72, 88, 122,and 139, and Che-2 Expression

[0225] Endogenous expression of a gene of the invention can also bemodulated by inactivating the endogenous gene or its promoter usingtargeted homologous recombination (see, e.g., U.S. Pat. No. 5,464,764).For example, a mutant, non-functional gene of the invention (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous gene of the invention (either the coding regions orregulatory regions of the gene of the invention) can be used, with orwithout a selectable marker and/or a negative selectable marker, totransfect cells that express the endogenous gene of the invention invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the gene of the invention.Such approaches are particularly suited for use in the agriculturalfield where modifications to ES (embryonic stem) cells can be used togenerate animal offspring with an inactive gene of the invention.However, this approach can be adapted for use in humans, provided therecombinant DNA constructs are directly administered or targeted to therequired site in vivo using appropriate viral vectors.

[0226] Alternatively, endogenous expression of a gene of the inventioncan be modulated by targeting deoxyribonucleotide sequencescomplementary to the regulatory region of the gene of the invention(i.e., the promoter and/or enhancers of a gene of the invention) to formtriple helical structures that prevent transcription of the gene of theinvention in target cells in the body (Helene, Anticancer Drug Res.6:569, 1981; Helene et al., Ann. N. Y Acad. Sci. 660:27, 1992; andMaher, Bioassays 14:807, 1992).

[0227] The invention includes methods for preparing pharmaceuticalcompositions for modulating the expression or activity of a polypeptideor nucleic acid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent that modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

[0228] The agent that modulates expression or activity can, for example,be a small molecule. For example, such small molecules include peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

[0229] It is understood that appropriate doses of small molecule agentsand protein or polypeptide agents depends upon a number of factorswithin the ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of these agents will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the agent to have upon the nucleic acid orpolypeptide of the invention. Examples of doses of a small moleculeinclude milligram or microgram amounts per kilogram of subject or sampleweight (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). Examples of doses of a protein or polypeptide include gram,milligram or microgram amounts per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 5 grams per kilogram,about 100 micrograms per kilogram to about 500 milligrams per kilogram,or about 1 milligram per kilogram to about 50 milligrams per kilogram).For antibodies, examples of dosages are from about 0.1 milligram perkilogram to 100 milligrams per kilogram of body weight (generally 10milligrams per kilogram to 20 milligrams per kilogram). If the antibodyis to act in the brain, a dosage of 50 milligrams per kilogram to 100milligrams per kilogram is usually appropriate. It is furthermoreunderstood that appropriate doses of one of these agents depend upon thepotency of the agent with respect to the expression or activity to bemodulated. Such appropriate doses can be determined using the assaysdescribed herein. When one or more of these agents is to be administeredto an animal (e.g., a human) in order to modulate expression or activityof a polypeptide or nucleic acid of the invention, a physician,veterinarian, or researcher can, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. In addition, it is understood that the specificdose level for any particular animal subject will depend upon a varietyof factors including the activity of the specific agent employed, theage, body weight, general health, gender, and diet of the subject, thetime of administration, the route of administration, the rate ofexcretion, any drug combination, and the degree of expression oractivity to be modulated.

[0230] As an alternative to making determinations based on the absoluteexpression level of selected genes, determinations may be based on thenormalized expression levels of these genes. Expression levels arenormalized by correcting the absolute expression level of a geneencoding a polypeptide of the invention by comparing its expression tothe expression of a different gene, e.g., a housekeeping gene that isconstitutively expressed. Suitable genes for normalization includehousekeeping genes such as the actin gene. This normalization allows thecomparison of the expression level in one sample (e.g., a patientsample), to another sample, or between samples from different sources.

[0231] Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a gene,the level of expression of the gene is determined for or more samples ofdifferent endothelial (e.g. intestinal endothelium, airway endothelium,or other mucosal epithelium) cell isolates, preferably 50 or moresamples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the gene(s) in question. Theexpression level of the gene determined for the test sample (absolutelevel of expression) is then divided by the mean expression valueobtained for that gene. This provides a relative expression level andaids in identifying extreme cases of disorders associated with aberrantexpression of a gene encoding a polypeptide of the invention protein orwith aberrant expression of a ligand thereof.

[0232] Preferably, the samples used in the baseline determination willbe from either or both of cells which aberrantly express a gene encodinga polypeptide of the invention or a ligand thereof (i.e. ‘diseasedcells’) and cells which express a gene encoding a polypeptide of theinvention at a normal levelor a ligand thereof (i.e. ‘normal’ cells).The choice of the cell source is dependent on the use of the relativeexpression level. Using expression found in normal tissues as a meanexpression score aids in validating whether aberrance in expression of agene encoding a polypeptide of the invention occurs specifically indiseased cells. Such a use is particularly important in identifyingwhether a gene encoding a polypeptide of the invention can serve as atarget gene. In addition, as more data is accumulated, the meanexpression value can be revised, providing improved relative expressionvalues based on accumulated data. Expression data from endothelial cells(e.g. mucosal endothelial cells) provides a means for grading theseverity of the disorder.

[0233] Detecting Proteins Associated with IFT 20, 27, 46, 52, 57, 72,88, 122, or 139, or Che-2

[0234] The invention also features polypeptides that interact with(e.g., bind directly or indirectly) IFT 20, 27, 46, 52, 57, 72, 88, 122,or 139, or Che-2. Any method suitable for detecting protein-proteininteractions may be employed for identifying transmembrane proteins,intracellular, or extracellular proteins that interact with polypeptidesof the invention. Among the traditional methods which may be employedare co-immunoprecipitation, cross-linking and co-purification throughgradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates and the use of polypeptides of the inventionto identify proteins in the lysate that interact with polypeptides ofthe invention. For these assays, the polypeptide of the invention can befull length polypeptide of the invention, a soluble extracellular domainof a polypeptide of the invention, or some other suitable polypeptide ofthe invention. Once isolated, such an interacting protein can beidentified and cloned and then used, in conjunction with standardtechniques, to identify proteins with which it interacts. For example,at least a portion of the amino acid sequence of a protein whichinteracts with the polypeptide of the invention can be ascertained usingtechniques well known to those of skill in the art, such as via theEdman degradation technique. The amino acid sequence obtained may beused as a guide for the generation of oligonucleotide mixtures that canbe used to screen for gene sequences encoding the interacting protein.Screening may be accomplished, for example, by standard hybridization orPCR techniques. Techniques for the generation of oligonucleotidemixtures and the screening are well-known. (Ausubel, supra; and “PCRProtocols: A Guide to Methods and Applications,” Innis et al., eds.Academic Press, Inc., NY, 1990).

[0235] Additionally, methods may be employed which result directly inthe identification of genes which encode proteins which interact withpolypeptides of the invention. These methods include, for example,screening expression libraries, in a manner similar to the well knowntechnique of antibody probing of λgt11 libraries, using labeledpolypeptide of the invention or a fusion protein of the invention, e.g.,a polypeptide of the invention or domain thereof fused to a marker suchas an enzyme, fluorescent dye, a luminescent protein, or to an IgFcdomain.

[0236] There are also methods capable of detecting protein interaction.A method that detects protein interactions in vivo is the two-hybridsystem (Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578, 1991). A kitfor practicing this method is available from Clontech (Palo Alto,Calif.).

[0237] III. Uses

[0238] A. Human and Animal Therapeutic Uses

[0239] Inhibition of IFT Particle Protein Function (Spermiogenesis andContraception) The sperm flagellum consists of a 9+2 axoneme whichgenerates sperm movement. In addition, the flagellum of the mammaliansperm contains accessory structures, viz. the fibrous sheath and theouter dense fibers, which are believed to stiffen the flagellum andenable it to finction in the viscous environment of the femalereproductive tract (Witman, Introduction to cilia and flagella. In:Ciliary and Flagellar Membranes (ed. R. A. Bloodgood). Plenum Press, NY.pp. 1-30, 1990). During spermiogenesis in the mammalian testis, a 9+2axoneme is formed within a flagellar membrane; as in other flagella,proper assembly of this axoneme must require IFT to move the axonemalprecursors to the tip of the flagellum, where they are assembled. Afterassembly of the axoneme, the accessory structures are assembledbeginning at the distal tip of the flagellum and progressing toward itsbase (Oko and Clermont, Mammalian spermatozoa: structure and assembly ofthe tail. In: Controls of Sperm Motility: Biological and ClinicalAspects. (ed. C. Gagnon), CRC Press, Boca Raton, Fla., pp. 3-27, 1990).Concomitant with this assembly, accessory structure proteins, previouslypresent in the cytoplasm of the spermiogenic cells, are mobilized fromthe cytoplasm to the flagellum. This transport of outer dense fiber andfibrous sheath precursors from the cytoplasm to the tip of the formingflagellum also must be dependent on IFT. Indeed, in the Tg737insertional mutant mouse, which has a defect in IFT, the sperm flagellumis not formed, and accessory structure precursors remain in the cellbody (J. San Agustin, G. Pazour and G. Witman, unpublished results).Therefore, in humans, a defect in IFT would be expected to result inazoospermia and oligozoospermia. Azoospermia and oligospermia are amongthe most common reasons for infertility in men. It is likely that agenetic defect in an IFT protein is responsible for at least some casesof azoospermia and oligozoospermia.

[0240] Because IFT is essential for sperm flagella formation, a drugthat inhibits IFT would block spermiogenesis and prevent fertility.Ideally, this drug would 1) target an IFT protein or isoform expressedspecifically in the testis, and 2) be able to cross the blood/testisbarrier. Such a drug, which can be administered to men orally or by anyother preferred route, would be specific for IFT in the testis and canserve as a male contraceptive.

[0241] Human and Animal Parasites

[0242] Many human and animal eukaryotic parasites have cilia or flagellathat are critical for their ability to infect and survive in theirhosts. Therefore, these parasites are likely to be susceptible toanti-IFT drugs that would block formation and function of their cilia orflagella. Ideally, anti-IFT drugs would be tailored to bind specificallyto the parasite's IFT proteins in order to avoid undesirable effects onthe host's cilia and flagella. However, because short-term drug-inducedinhibition of IFT in humans and animals is unlikely to causelong-lasting side effects, the host most likely can tolerate treatmentwith non-host specific anti-IFT drugs for the short time periodsnecessary to eliminate a parasite. Some examples of ciliated orflagellated parasites that would be amenable to treatment by anti-IFTdrugs are given below.

[0243] Parasitic Protozoa

[0244] Parasitic protozoa cause numerous human and animal diseases,including malaria, African sleeping sickness, trypanosomosis,leishmanioses, trichomonosis, and giardiosis. All of these parasiteshave stages of their life cycles that are dependent on cilia orflagella, and in many cases cilia or flagella are present during thoselife-cycle stages that occur in the human or animal host, making theparasites vulnerable to drugs which would block IFT and hence inhibitcilia or flagella assembly or function.

[0245] One example is Giardia sp., an intestinal parasite which causesdebilitating diarrhea and other symptoms in humans. Giardia liveprimarily in the upper portion of the lumen of the small intestine,where they attach to the enterocytes of the intestinal wall (Marquardtet al., Parasitology and Vector Biology, 2nd ed., Academic Press, SanDiego, 2000). Each Giardia cell has 8 motile flagella, which presumablyare used to stay in the upper portion of the small intestine and toreach the site of attachment. An anti-IFT drug, taken orally, wouldinhibit assembly of flagella in newly divided Giardia and causedisassembly of previously formed flagella in non-dividing Giardia. As aresult, the Giardia would not be able to move up the lumen of the smallintestine, or reach the wall of the small intestine to attach to itssurface. In the absence of motility and anchorage, the Giardia would bepassed out of the intestine and the infection would be eliminated.

[0246] An anti-IFT drug also would be effective against trypanosomes,which are responsible for diseases such as African sleeping sickness andChagas' disease in humans, and nagana in cattle. Trypanosomes circulatein the blood where they reproduce by asexual division. These parasitesare characterized by the presence of a single motile flagellum thatarises from a flagellar pocket and is enclosed by a sheath called theundulating membrane. An anti-IFT drug would block assembly of theflagellum and the flagellar sheath and affect the trypanosome's lifecycle and host-parasite interactions in at least three ways. First, byblocking flagellar assembly, it would affect those normal life processesthat are dependent upon flagellar motility. Second, the ability of thetrypanosome to evade the human or animal host's defense mechanisms inthe bloodstream is dependent on its production of a protectiveglycoprotein coat that covers both the cell body and the flagellarsheath (Marquandt et al., 1999). Movement of these proteins onto theflagellar sheath is dependent upon IFT (Rosenbaum and Witman, 2001;Bloodgood, 2000). In the absence of IFT, the protective coating wouldnot be present on the flagellar sheath and the parasite would besusceptible to attack by the host immune system. Third, the flagellum isessential for the trypanosome's attachment to, and infection of, theinsect vector (e.g., tsetse fly, kissing bugs) which take up theparasite from the human or animal bloodstream and then spread thedisease to other humans or animals (Vickerman and Tetley, Flagellarsurfaces of parasitic protozoa and their role in attachment, In: Ciliaryand Flagellar Membranes (ed. R. A. Bloodgood), Plenum Press, N.Y., 1989;Marquardt et al., Parasitology and Vector Biology, 2nd ed., AcademicPress, San Diego, 2000). By eliminating the trypanosome's flagellumprior to uptake of the parasite by the insect vector, the trypanosome'slife cycle would be interrupted and transmission of the parasite to newhosts would be prevented. Because trypanosomes live in the bloodstream,they would be very susceptible to anti-IFT drugs administered byintravenous injection.

[0247] Trichomonads are flagellated parasitic protozoans that likewisecan be treated with an anti-IFT drug. Tritrichomonas foetus causestrichomonad abortion in cattle and other bovines. In the United Statesalone, it is estimated that there is an annual loss of $650 million tothe cattle industry from trichomonosis (Marquardt et al., Parasitologyand Vector Biology, 2nd ed., Academic Press, San Diego, 2000). T. foetusinfects the reproductive tracts of both cows and bulls. It reproduces byasexual division, and is spread by sexual intercourse. Upon introductioninto the reproductive tract of a cow during sexual intercourse, thetrichomonads reproduce in the vagina. If the animal becomes pregnant,the organisms may invade the uterus and infect the developing fetus,causing abortion (Marquardt et al., Parasitology and Vector Biology, 2nded., Academic Press, San Diego, 2000). Trichomonads have three or moremotile anterior flagella, and a motile recurrent flagellum usuallyattached to the body by an undulating membrane. These flagellapresumably are essential for movement of the parasite up thereproductive tract. Treatment of infected cows with an anti-IFT drugdelivered orally or by intravagina suppository would prevent assembly ofthe flagella of newly divided cells, and result in loss of flagella ofnon-dividing cells. In the absence of the flagellum, the trichomonadswould be immotile and would be unable to move up the female reproductivetract and cause abortion. Moreover, rendering the parasites immotile andparalyzed would decrease their ability to withstand the host's ownimmune defenses, so that the infection can be completely eliminated.Similarly, treatment of bulls with anti-IFT drugs administered orallywould render the trichomonads immotile, so they can be eliminated by thebull's own immune system.

[0248]Trichomonas vaginalis causes trichomonas vaginitis in humans andcan be similarly treated to eliminate infection.

[0249] Parasitic Platyhelminthes

[0250] Many human and animal parasites are members of the phylumPlatyhelminthes. Parasitic Platyhelminthes include liver flukes,intestinal flukes, lung flukes and other trematodes, and cestodes ortapeworms. In humans, these parasites cause severe illnesses such asschistosomosis, dicrocoeliosis, clonorchiasis, opisthorchosis,echinostomatiasis, heterophyidosis, swimmer's itch, taeniosis, andcysticercosis. Infection of livestock by these parasites results in hugeeconomic losses in cattle- and sheep-raising areas. All Platyhelmintheshave an excretory system based on the flame cell or protonephridium, inwhich currents are created by a tuft of vigorously beating cilia(Bogitsh and Cheng, Human Parisitology, Academic Press, San Diego, 1979;Marquardt et al., Parasitology and Vector Biology, 2nd ed., AcademicPress, San Diego, 2000). The flame cell cilia may have additionalsensory or osmoregulatory functions. In addition, some parasiticPlatyhelminthes, such as flatworms, have external cilia that arebelieved to have a sensory role (Marquardt et al., Parasitology andVector Biology, 2nd ed., Academic Press, San Diego, 2000). Because allcilia and flagella appear to be dependent upon IFT for their assemblyand maintenance, one would expect that a drug that inhibited IFT wouldprevent assembly of the cilia, or result in disassembly of previouslyformed cilia, in these parasites, causing malfunction of the parasite'sosmoregulatory and/or nervous systems. Thus, treatment of infectedhumans or animals with an anti-IFT drug would control or cure infectionsby these Platyhelminthes. The anti-IFT drug would be administered orallyor intravenously, depending on the site of infection.

[0251] Parasitic Nematodes

[0252] Over half of the world's population is estimated to be infectedby at least one or more species of parasitic nematodes. Animal parasiticnematodes are also widespread and exhibit a wide diversity. Nematodessense their environment with a set of sensory neurons in which thedendrites are ciliated. These sensory cilia extend outside of the toughnematode cuticle and are exposed to the outer environment where they areresponsible for detecting various chemical signals as well as monitorosmotic conditions. Nematodes, for example, will move away from highsalt conditions so as to protect themselves from osmotic stress. Innematode mutants where the sensory cilia are structurally defective, theorganism is unable to identify dangerous levels of salt; these mutantsare also defective in chemosensation (Perkins et al., Dev. Biol.,117:456-487, 1986). Since IFT is required for the formation andmaintenance of these sensory cilia, blocking IFT with various anti-IFTagents should result in nematodes that have lost their ability to sensetheir environment. This will adversely affect the organism's ability todetermine where it is going and in finding sexual partners. As describedin detail under Combating phytopathogenic nematodes, nematode-specificRNA interference should also work in combating human and animal nematodeparasites.

[0253] Ascariasis, caused by Ascaris lumbricoides is globallydistributed with more than 1.4 billion persons infected throughout theworld (Khuroo, Gastroenterol Clin North Am, 25:553577 1996). Ascarisinfection begins with the ingestion of infective eggs. Once in the smallintestine, the eggs hatch and the resulting juveniles pass through theintestinal wall and enter the bloodstream and eventually end up in thelungs. After entering the airspace, Ascaris move up into the pharynxwhere they are swallowed and reappear in the small intestine. The adultfemale can lay as many as 200,000 eggs a day in the small intestine.Potentially fatal pathologies includes ascariasis pneumonia due topulmonary hemorrhaging and inflammation and physical blockage of thegastrointestinal tract due to a large mass of the Ascaris nematodes.Ascaris suum is a common pig parasite that also infects humans but thejuvenile gets no farther than the lungs; pathology includes ascariasispneumonia. In the intestine, Ascaris spp can be susceptible to orallyadministered anti-IFT agents. Anti-IFT agents can also be administeredby intra-venous injection to treat juvenile nematodes in thebloodstream.

[0254] The nematodes known as hookworms infect over a billion people.Most of these infections are caused by either Ancylostoma duodenale orNecator americanus. The life cycle of hookworms is similar to that ofAscaris and suggests that anti-IFT treatments that are successful intreating ascariasis should also be successful in treating hookworminfections.

[0255] The nematodes known as whipworms are also estimated to infectover a billion people. These infections, caused by Trichuris trichiura,are typically confined to the large intestine. Anti-IFT agents can beadministered either orally or by suppository.

[0256] Animals are infected at a high rate by a large and diverse groupof nematodes. In all cases, however, the nematodes may be susceptible toanti-IFT treatments described above. These include nematode-specific RNAinterference directed against expression of IFT subunits. Theappropriate IFT-derived double stranded RNA can be administered directlyto the infected animals or used as a preventive treatment prior toingestion of the infective eggs. Alternatively, transgenic animalstrains can be developed that contain stable transformed DNA thatencodes double stranded RNA derived from nematode-specific sequencesthat encode IFT proteins. In this way, entire populations of livestockcan be produced that will no longer support infection by one or morespecies of parasitic nematode. Two obvious benefits of this approach are(1) that animals will not have to be periodically treated to reduce orremove infection and (2) it may be possible to eradicate specificinfectious species by making all hosts resistant.

[0257] Restoration of IFT Particle Protein Function

[0258] Gene Therapy The gene constructs of the invention can also beused as a part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of a polypeptide ofthe invention. The invention features expression vectors for in vivotransfection and expression of a polypeptide of the invention inparticular cell types so as to reconstitute the function of, oralternatively, antagonize the function of a polypeptide of the inventionin a cell in which that polypeptide is misexpressed. Expressionconstructs of polypeptides of the invention, may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering a gene of the invention to cells invivo. Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,and herpes simplex virus- 1, or recombinant bacterial or eukaryoticplasmids. Viral vectors transfect cells directly; plasmid DNA can bedelivered with the help of, for example, cationic liposomes (lipofectin)or derivatized (e.g. antibody conjugated), polylysine conjugates,gramacidin S, artificial viral envelopes or other such intracellularcarriers, as well as direct injection of the gene construct or CaPO₄precipitation carried out in vivo.

[0259] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA, encoding a polypeptide of the invention. Infection of cells with aviral vector has the advantage that a large proportion of the targetedcells can receive the nucleic acid. Additionally, molecules encodedwithin the viral vector, e.g., by a cDNA contained in the viral vector,are expressed efficiently in cells which have taken up viral vectornucleic acid.

[0260] Retrovirus vectors and adeno-associated virus vectors can be usedas a recombinant gene delivery system for the transfer of exogenousgenes in vivo, particularly into humans. These vectors provide efficientdelivery of genes into cells, and the transferred nucleic acids arestably integrated into the chromosomal DNA of the host. The developmentof specialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). A replication defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include yCrip, yCre, y2 and yAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:30143018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0261] Another viral gene delivery system useful in the presentinvention utilizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See, for example, Berkneret al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells and can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al. (1992) cited supra).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis in situwhere introduced DNA becomes integrated into the host genome (e.g.,retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large (up to 8 kilobases) relative to othergene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham(1986) J. Virol. 57:267).

[0262] Yet another viral vector system useful for delivery of thesubject gene is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. (1992) Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. BioL 4:20722081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0263] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of apolypeptide of the invention in the tissue of an animal. Most nonviralmethods of gene transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inpreferred embodiments, non-viral gene delivery systems of the presentinvention rely on endocytic pathways for the uptake of the subject geneof the invention by the targeted cell. Exemplary gene delivery systemsof this type include liposomal derived systems, poly-lysine conjugates,and artificial viral envelopes. Other embodiments include plasmidinjection systems such as are described in Meuli et al. (2001) J. InvestDermatol. 116(l):131-135; Cohen et al. (2000) Gene Ther 7(22):1896-905;or Tam et al. (2000) Gene Ther 7(21):1867-74.

[0264] In a representative embodiment, a gene encoding a polypeptide ofthe invention can be entrapped in liposomes bearing positive charges ontheir surface (e.g., lipofectins) and (optionally) which are tagged withantibodies against cell surface antigens of the target tissue (Mizuno etal. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;Japanese patent application 1047381; and European patent publicationEP-A-43075).

[0265] In clinical settings, the gene delivery systems for thetherapeutic gene of the invention can be introduced into a patient byany of a number of methods, each of which is familiar in the art. Forinstance, a pharmaceutical preparation of the gene delivery system canbe introduced systemically, e.g. by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Patent 5,328,470) or by stereotacticinjection (e.g. Chen et al. (1994) PNAS 91: 3054-3057).

[0266] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced in tact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0267] Compounds That Affect the Activity of IFT

[0268] Drugs that stop IFT can have important anti-fertility,anti-parasitic, and anti-pesticide activities while drugs that enhanceIFT can be used to improve ciliary function in patients with diseasescaused by reduced IFT. In vitro binding assays can be used to identifycompounds that bind with high affinity to IFT particle proteins. Theidentified compounds can then be used in in vivo assays to determine ifthey affect ciliary assembly. Compounds may include small organic orinorganic molecules, peptides, peptidomimetics, nucleic acids, orcarbohydrates.

[0269] Genes encoding IFT particle proteins from humans or other speciescan be expressed in bacterial, mammalian, insect, or other cells andpurified. These proteins can then be used in high-throughput screens ofchemical compounds to identify those that bind, e.g., with highaffinity. The identified compounds can then be screened to determine ifthey affect ciliary assembly.

[0270] The effect of the drugs on ciliary assembly can be determined byadding them to cultures of ciliated organisms and observing how thisaffects the cilia. For example, the effect on trypanosome ciliaryassembly can be determined by adding the compounds to axenic culturesand observing the cells by light microscopy to see if ciliary assemblyis affected (Tyler and Engman, Cell Motil Cytoskeleton, 46(4):269-78,2000). The effect on nemotode sensory cilia can be determined byassaying the ability of treated worms to take up membrane-solublefluorescent dyes. Nematodes that lack cilia do not take up the dyes(Starich et al, Genetics, 139:171-188, 1995). The effect on mammalianprimary cilia assembly can be determined by adding the compounds tocultured mammalian cells and assaying ciliary assembly byimmunofluorescence microscopy (Wheatley et al., Cell Biol. Int.,20:73-81, 1996).

[0271] Compounds that bind to IFT particle proteins can also be screenedto determine if they enhance IFT. This can be done using cultured cellsderived from the Tg737 mutant mouse (Taulman et al., Mol Biol Cell,12(3):589-99, 2001). This mouse has a reduced amount of the Tg737/IFT88protein (Taulman et al., Mol Biol Cell, 12(3):589-99, 2001; Pazourunpublished observation) and shows defects in assembly of kidney primarycilia (Pazour et al., Mol. Biol. Cell, 151:709-718, 2000). Presumablythe cells derived from this mouse will also show defects in ciliaryassembly and can be used to screen for compounds that enhance IFT tocompensate for the reduced amount of protein.

[0272] Assays for Compounds Capable of Restoring or Inhibiting IFTFunction

[0273] Even after a particular intracellular target is selected, themeans by which new IFT protein function restoration agents areidentified pose certain challenges. Despite the increased use ofrational drug design, a preferred method continues to be the massscreening of compound “libraries” for active agents by exposing culturesof cells with cilia or flagella to the test compounds and assaying forinhibition or restoration of normal IFT protein activity. In testingthousands or tens of thousands of compounds, however, a correspondinglylarge number of cell cultures of interest must be grown over timeperiods which are relatively long. Moreover, a compound that is found toinhibit or restore normal IFT protein function in culture may be actingnot on the desired target but on a different, less unique component ofthe IFT system, with the result that the compound may act against hostcells as well and thereby produce unacceptable side effects.Consequently, there is a need for an assay or screening method that morespecifically identifies those agents that are active against a certainintracellular target.

[0274] Additionally, there is a need for assay methods having greaterthroughput, that is, assay methods that reduce the time and materialsneeded to test each compound of interest.

[0275] The invention provides methods for identifying agents capable ofmodululating, e.g., restoring or inhibiting, IFT function. In somemethods, screening for potential or candidate IFT function restorativeor inhibitory agents is accomplished by identifying those compounds(e.g., small organic molecules) that interact with, e.g., bind directlyor indirectly, to an IFT polypeptide and/or inhibit the activity of aIFT polypeptide or the expression of an IFT gene. For example, screenscan be performed that identify those compounds that inhibit an IFTactivity described herein.

[0276] In various suitable methods, screening for anti-IFT agents can beaccomplished by (i) identifying those compounds that bind to IFT (andare thus candidate anti-IFT compounds) and (ii) further testing suchcandidate compounds for their ability to inhibit intraflagellartransport in vitro or in vivo, in which case they are anti-IFT agents.

[0277] Specific binding of a test compound to a polypeptide can bedetected, for example, in vitro by reversibly or irreversiblyimmobilizing the test compound(s) on a substrate, e.g., the surface of awell of a 96-well polystyrene microtitre plate. Methods for immobilizing25 polypeptides and other small molecules are well known in the art. Forexample, the microtitre plates can be coated with a IFT polypeptide byadding the polypeptide in a solution (typically, at a concentration of0.05 to 1 mg/ml in a volume of 1-100 ml) to each well, and incubatingthe plates at room temperature to 37° C. for 0.1 to 36 hours.Polypeptides that are not bound to the plate can be removed by shakingthe excess solution from the plate, and then washing the plate (once orrepeatedly) with water or a buffer. Typically, the polypeptide is inwater or a buffer.

[0278] The plate is then washed with a buffer that lacks the boundpolypeptide. To block the free protein-binding sites on the plates, theplates are blocked with a protein that is unrelated to the boundpolypeptide. For example, 300 ml of bovine serum albumin (BSA) at aconcentration of 2 mg/ml in Tris-HCl is suitable. Suitable substratesinclude those substrates that contain a defined cross-linking chemistry(e.g., plastic substrates, such as polystyrene, styrene, orpolypropylene substrates from Coming Costar Corp., Cambridge, Mass., forexample). If desired, a beaded particle, e.g., beaded agarose or beadedSepharose, can be used as the substrate. The IFT is then added to thecoated plate and allowed to bind to the test compound (e.g., at 37° C.for 0.5-12 hours). The plate then is rinsed as described above.

[0279] Binding of the test compound to the IFT can be detected by any ofa variety of known methods. For example, an antibody that specificallybinds to a IFT polypeptide can be used in an immunoassay. If desired,the antibody can be labeled (e.g., fluorescently or with a radioisotope)and detected directly (see, e.g., West and McMahon, J. Cell Biol.74:264, 1977). Alternatively, a second antibody can be used fordetection (e.g., a labeled antibody that binds to the Fc portion of ananti-YphC antibody). In an alternative detection method, the IFTpolypeptide is labeled, and the label is detected (e.g., by labeling aIFT polypeptide with a radioisotope, fluorophore, chromophore, or thelike). In still another method, the IFT polypeptide is produced as afusion protein with a protein that can be detected optically, e.g.,using green fluorescent protein (which can be detected under UV light).In an alternative method, the polypeptide can be produced as a fusionprotein with an enzyme having a detectable enzymatic activity, such ashorseradish peroxidase, alkaline phosphatase, J-galactosidase, orglucose oxidase. Genes encoding all of these enzymes have been clonedand are available for use by those of skill in the art. If desired, thefusion protein can include an antigen, and such an antigen can bedetected and measured with a polyclonal or monoclonal antibody usingconventional methods. Suitable antigens include enzymes (e.g.,horseradish peroxidase, alkaline phosphatase, and J-galactosidase) andnon-enzymatic polypeptides (e.g., serum proteins, such as BSA andglobulins, and milk proteins, such as caseins).

[0280] In various in vivo methods for identifying polypeptides that bindto IFT, the conventional two-hybrid assays of protein/proteininteractions can be used (see e.g., Chien et al., Proc. Natl. Acad. Sci.USA, 88:9578, 1991; Fields et al., U.S. Pat. No. 5,283,173; Fields andSong, Nature, 340:245, 1989; Le Douarin et al., Nucleic Acids Research,23:876, 1995; Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10315-10320,1996; and White, Proc. Natl. Acad. Sci. USA, 93:1000107917-145001 10003,1996). Generally, the two-hybrid methods involve in vivo reconstitutionof two separable domains of a transcription factor. One fusion proteincontains the IFT polypeptide fused to either a transactivator domain orDNA binding domain of a transcription factor (e.g., of Gal4). The otherfusion protein contains a test polypeptide fused to either the DNAbinding domain or a transactivator domain of a transcription factor.Once brought together in a single cell (e.g., a yeast cell or mammaliancell), one of the fusion proteins contains the transactivator domain andthe other fusion protein contains the DNA binding domain. Therefore,binding of the IFT polypeptide to the test polypeptide (i.e., candidateanti-IFT agent) reconstitutes the transcription factor. Reconstitutionof the transcription factor can be detected by detecting expression of agene (i.e., a reporter gene) that is operably linked to a DNA sequencethat is bound by the DNA binding domain of the transcription factor.Kits for practicing various two-hybrid methods are commerciallyavailable (e.g., from Clontech; Palo Alto, Calif.).

[0281] The methods described above can be used for high throughputscreening of numerous test compounds to identify candidate compoundsthat modulate the function or activity of IFT particle proteins. Havingidentified a test compound as a candidate compound, the candidatecompound can be further tested for inhibition of intraflagellartransport in vitro or in vivo (e.g., using a cell, animal, e.g., rodent,model ) if desired.

[0282] In vitro, further testing can be accomplished by means known tothose in the art such as an enzyme inhibition assay or a whole-cellgrowth inhibition assay. For example, an agar dilution assay identifiesa substance that inhibits cell growth. Microtiter plates are preparedwith serial dilutions of the test compound, adding to the preparation agiven amount of growth substrate, and providing a preparation of cells.Inhibition of cell growth is determined, for example, by observingchanges in optical densities of the cell cultures.

[0283] Inhibition of cell growth is demonstrated, for example, bycomparing (in the presence and absence of a test compound) the rate ofgrowth or the absolute growth of cells. Inhibition includes a reductionin the rate of growth or absolute growth by at least 20%. Particularlypotent test compounds can further reduce the growth rate (e.g., by atleast 25%, 30%, 40%, 50%, 75%, 80%, or 90%).

[0284] Animal (e.g., rodent such as murine) models of intraflagellartransport are known to those of skill in the art, and such animal modelsystems are acceptable for screening potential compounds capable ofrestoring intraflagellar transport function as an indication of theirtherapeutic efficacy in human patients. In a typical in vivo assay, ananimal is treated with a compound that is capable of restoring IFTfunction, and conventional methods and criteria are used to diagnose themammal as having impaired intraflagellar transport. The candidate IFTrestorative agent then is administered to the mammal at a dosage of1-100 mg/kg of body weight, and the mammal is monitored for signs of IFTfunction restoration. Of course, the results obtained in the presence ofthe test compound should be compared with results in control animals,which are not treated with the test compound. Administration ofcandidate IFT function restorative agents to the mammal can be carriedout as described below, for example.

[0285] IFT function restorative agents can be identified with highthroughput assays to detect IFT activity, e.g., the ability to growcilia or flagella. For example, this restoration can be effected bysmall molecules binding directly to the IFT particle protein, or bybinding of small molecules to other essential polypeptides in abiochemical pathway in which IFT participates.

[0286] The invention also provides methods of identifying agents (suchas compounds, other substances, or compositions) that affect, orselectively affect, (such as inhibit, restore or otherwise modify) theactivity of and/or expression of IFT particle polypeptides (or the IFTparticle itself), by contacting an IFT polypeptide or the nucleotidesequence encoding the same with the agent and then measuring theactivity of IFT, e.g., intraflagellar transport. In a related aspect,the invention features a method of identifying agents (such ascompounds, other substances or compositions comprising same) that affect(such as inhibit, restore, or otherwise modify) the activity of and/orexpression of nucleic acids encoding IFT particle polypeptides, bymeasuring the activity of and/or expression of IFT in the presence ofthe agent or after the addition of the agent in: (a) a cell line intowhich has been incorporated a recombinant construct including thenucleotide sequence of the nucleic acid encoding an IFT polypeptide oran allelic variation thereof; or (b) a cell population or cell line thatnaturally selectively expresses IFT polypeptides, and then measuring theactivity of IFT polypeptides (or intraflagellar transport as a whole)and/or the expression thereof.

[0287] Since the IFT nucleic acids described herein have beenidentified, they can be cloned into various host cells (e.g., fungi, E.coli, or yeast) for carrying out such assays in whole cells.

[0288] To identify compounds that modulate expression of the IFT genethe test compound(s) can be added at varying concentrations to theculture medium of cells. Such test compounds can include small molecules(typically, non-protein, non-polysaccharide chemical entities),polypeptides, and nucleic acids. The expression of IFT nucleic acid isthen measured, for example, by Northern blot PCR analysis or RNAseprotection analyses using a nucleic acid molecule of the invention as aprobe. The level of expression in the presence of the test molecule,compared with the level of expression in its absence, will indicatewhether or not the test molecule alters the expression of IFT. BecauseIFT is essential for survival of many organisms (e.g., mammals), testcompounds that inhibit the expression and/or function of IFT nucleicacids are expected inhibit growth of, or kill, the cells that expressIFT.

[0289] Some specific embodiments of the present invention relate toassay methods for the identification of anti-IFT agents using assays foranti-IFT agents which may be carried out both in whole cell preparationsand in ex vivo cell-free systems. In each instance, the assay target isthe IFT nucleotide sequence and/or the IFT polypeptide. Test compoundswhich are found to inhibit the IFT nucleotide sequence and/or IFTpolypeptide in any assay method of the present invention are thusidentified as potential or candidate anti-IFT agents. It is expectedthat the assay methods of the present invention will be suitable forboth small and large-scale screening of test compounds as well as inquantitative assays such as serial dilution studies where the target IFTnucleotide sequence or the IFT polypeptide are exposed to a range oftest compound concentrations.

[0290] A variety of protocols for detecting and measuring the expressionof IFT, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson IFT polypeptides is suitable; alternatively, a competitive bindingassay may be employed. These and other assays are described, among otherplaces, in Hampton, R et al. (1990, Serological Methods, A LaboratoryManual, APS Press, St Paul, Minn.) and Maddox, DE et al. (1983, J. Exp.Med. 158:121).

[0291] Pharmaceutical Formulations

[0292] The present invention also provides a pharmaceutical compositionfor treating an individual in need of such treatment of a disease causedby abnormal functioning of at least one IFT protein (or that can betreated by inhibiting abnormal IFT protein activity); the treatmentmethod entails administering a therapeutically effective amount of anagent that affects (such as inhibits) the activity and apharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0293] The pharmaceutical compositions can be used for humans or animalsand will typically include any one or more of a pharmaceuticallyacceptable diluent, carrier, excipient, or adjuvant. The choice ofpharmaceutical carrier, excipient, and diluent can be selected withregard to the intended route of administration and standardpharmaceutical practice. The pharmaceutical compositions can include as(or in addition to) the carrier, excipient, or diluent, any suitablebinder(s), lubricant(s), suspending agent(s), coating agent(s), orsolubilizing agent(s).

[0294] The invention includes pharmaceutical formulations that include apharmaceutically acceptable excipient and an anti-IFT agent identifiedusing the methods described herein. In particular, the inventionincludes pharmaceutical formulations that restore normal function to atleast one IFT protein. Such pharmaceutical formulations can be used in amethod of treating abnormal function of at least one IFT protein in anorganism. Such a method entails administering to the organism atherapeutically effective amount of the pharmaceutical formulation,i.e., an amount sufficient to ameliorate signs and/or symptoms ofabnormal IFT protein function. In particular, such pharmaceuticalformulations can be used to treat abnormal IFT protein function inmammals such as humans and domesticated mammals (e.g., cows, pigs, dogs,and cats), and in plants. The efficacy of such IFT protein functionrestoration agents in humans can be estimated in an animal model systemwell known to those of skill in the art (e.g., mouse systems of abnormalIFT protein function).

[0295] Treatment includes administering a pharmaceutically effectiveamount of a composition containing IFT protein function restorationagent to a subject in need of such treatment, thereby restoring normalIFT protein function in the subject. Such a composition typicallycontains from about 0.1 to 90% by weight (such as 1 to 20% or 1 to 1%)of a IFT protein function restoration agent of the invention in apharmaceutically acceptable carrier.

[0296] Solid formulations of the compositions for oral administrationcan contain suitable carriers or excipients, such as corn starch,gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, oralginic acid. Disintegrators that can be used include, withoutlimitation, micro-crystalline cellulose, corn starch, sodium starchglycolate and alginic acid. Tablet binders that can be used includeacacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone (Povidone®), hydroxypropyl methylcellulose,sucrose, starch, and ethylcellulose. Lubricants that can be used includemagnesium stearates, stearic acid, silicone fluid, talc, waxes, oils,and colloidal silica.

[0297] Liquid formulations of the compositions for oral administrationprepared in water or other aqueous vehicles can contain varioussuspending agents such as methylcellulose, alginates, tragacanth,pectin, kelgin, carageenan, acacia, polyvinylpyrrolidone, and polyvinylalcohol. The liquid formulations can also include solutions, emulsions,syrups and elixirs containing, together with the active compound(s),wetting agents, sweeteners, and coloring and flavoring agents. Variousliquid and powder formulations can be prepared by conventional methodsfor inhalation into the lungs of the mammal to be treated.

[0298] Injectable formulations of the compositions can contain variouscarriers such as vegetable oils, dimethylacetamide, dimethylformamide,ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injections, water soluble versions of the compounds canbe administered by the drip method, whereby a pharmaceutical formulationcontaining the anti-IFT agent and a physiologically acceptable excipientis infused. Physiologically acceptable excipients can include, forexample, 5% dextrose, 0.9% saline, Ringer's solution, or other suitableexcipients. For intramuscular preparations, a sterile formulation of asuitable soluble salt form of the compounds can be dissolved andadministered in a pharmaceutical excipient such as Water-for-Injection,0.9% saline, or 5% glucose solution. A suitable insoluble form of thecompound can be prepared and administered as a suspension in an aqueousbase or a pharmaceutically acceptable oil base, such as an ester of along chain fatty acid, (e.g., ethyl oleate).

[0299] A topical semi-solid ointment formulation typically contains aconcentration of the active ingredient from about 1 to 20%, e.g., 5 to10% in a carrier such as a pharmaceutical cream base. Variousformulations for topical use include drops, tinctures, lotions, creams,solutions, and ointments containing the active ingredient and varioussupports and vehicles.

[0300] The optimal percentage of the IFT protein function restorationagents in each pharmaceutical formulation varies according to theformulation itself and the therapeutic effect desired in the specificpathologies and correlated therapeutic regimens. Appropriate dosages ofthe IFT protein function restoration agents can be determined by thoseof ordinary skill in the art of medicine by monitoring the mammal forsigns of disease amelioration or inhibition, and increasing ordecreasing the dosage and/or frequency of treatment as desired. Theoptimal amount of the IFT protein function restoration agent used fortreatment of conditions caused by or contributed to by abnormal IFTprotein function depends upon the manner of administration, the age andthe body weight of the subject, and the condition of the subject to betreated. Generally, the IFT protein function restoration compound isadministered at a dosage of 1 to 100 mg/kg body weight, and typically ata dosage of 1 to 10 mg/kg body weight.

[0301] B. Diagnostic Uses

[0302] Diagnosis of Defective or Absent IFT Particle Proteins

[0303] PCR to Detect Missing or Defective IFT Particle Protein Genes

[0304] RNA is extracted from patient biopsy material, e.g. nasalscrapings, and reverse transcribed into cDNA. PCR reactions are carriedout on this cDNA using pairs of PCR primers designed from the sequenceof the human homologue of each of the IFT particle protein genes. If thepatient lacked mRNA for any of the genes, then no product would beamplified. The PCR products can also be analyzed by sequencing or otherstandard methods to identify nucleotide changes. The Tg737 mutant mousehas reduced levels of the mRNA derived from the mouse Tg737/IFT88 gene(Moyer et al., 1994).

[0305] Alternatively, PCR can be used to amplify exons of genes encodingIFT particle proteins from genomic DNA purified from patient blood.These amplified products can then be sequenced or analyzed by otherstandard methods to identify nucleotide changes.

[0306] Antibody Assays to Identify Missing IFT Proteins

[0307] Protein extracts would be prepared from patient biopsy material,e.g. nasal scrapings, and analyzed by enzyme-linked immunosorbent assay(ELISA) or by western blot analysis using antibodies to the IFT particleproteins. The amount of protein in the patient sample would be comparedto normal controls to determine if the patient lacks a particular IFTparticle protein. Chlamydomonas cells with a mutation in the IFT88 genelack the IFT88 protein and IFT57 protein (Pazour et al., 2000) and theTg737 mutant mouse has reduced amounts of full length Tg737/IFT88protein (Pazour unpublished; Taulman et al., 2001).

[0308] C. Use in Agriculture

[0309] Combating phytopathogenic nematodes

[0310] Phytopathogenic nematodes are responsible for tens of billions ofdollars lost each year for farmers throughout the world (Williamson,Curr Opin Plant Biol, 2:327-331, 1999). A new way to combat theseparasites is the use of internalizable agents that interfere with thenematode's ability to sense its environment by disrupting the structureand function of nematode sensory cilia. Nematodes largely sense theirenvironment by chemosensation, a process that occurs at the sensorycilia of sensory neurons (Troemel, Bioessays, 21:1011-1020, 1999). Lossof function of these cilia will have multiple effects, including theloss of ability to locate the host plant as well as reproductive mates.

[0311] Small Molecule Inhibitors of Intraflagellar Transport

[0312] Intraflagellar Transport (IFT) is necessary for both theconstruction and continued maintenance of cilia and flagella in diverseorganisms, including nematodes (Rosenbaum and Witman, unpublishedmanuscript, 2001). If IFT is interrupted, cilia shorten and becomedysfunctional. We propose to block IFT with one or more small molecules.Large numbers of small molecules will be screened for their ability todisrupt IFT and thus, sensory cilia function. The IFT motors, kinesin-IIand cytoplasmic dynein 1b would be likely targets but other IFTmachinery might also be involved. There is already precedence for thisstrategy. Monastrol is a small organic molecule that was found viascreening to selectively inhibit a subgroup of kinesins known as Eg5 orbimC (Mayer et al, Science, 286:971-974, 1999). EgS inhibition bymonastrol is specific and does not affect the behavior of otherkinesins.

[0313] The screening process would identify the loss of sensory ciliafunction. Normal nematode sensory cilia are exposed to the environmentand have the unique ability to take up membrane-soluble fluorescent dyessuch as DiO and DiI (Molecular Probes, Inc). The cilia-dependent uptakeof these dyes into the sensory neurons allows for easy and fastscreening using fluorescence microscopy and has been used to identify arelatively large number of sensory cilia mutants (Starich et al,Genetics, 139:171-188, 1995). In mutants where the sensory cilia arestructurally defective and fail to extend out into the environment, nodye is taken up into the sensory neurons. This same screen can be usedto identify agents that cause normal, full-length sensory cilia toshorten to the point at which they are no longer exposed to theenvironment. As for the anti-IFT agents, many possible small moleculescan be screened. Since monastrol binds and specifically inhibits Eg5kinesin, it would be logical to try variations of this molecule sinceslight changes in chemical structure can result in a molecule that bindsand inhibits kinesin-II. Molecules that affect nematode IFT would likelybe added topically to the plant or the soil. Another, more specificapproach involves identification of small peptide(s) that block IFT.

[0314] Identification of one or more small peptides that block nematodeIFT is particularly attractive because host plants can be transformedwith the appropriate vector so that the host plant makes the inhibitorypeptide(s). Transformation would be mediated by Agrobacteriumtumefaciens with a standard vector such as pCAMBIA1380 or pCAMBIA1390(Center for the Application of Molecular Biology to InternationalAgriculture) encoding the inhibitory peptide. Different promoters can bechosen which would result in either constitutive expression ortissue-specific expression of the peptide gene. For example, manynematodes attack their host plant at the roots. Root-specificexpression, therefore, is desirable.

[0315] RNA Interference of Nematode IFT

[0316] Known as RNA interference or RNAi, the introduction ofgene-specific sequences of double-stranded RNA (dsRNA) result in aspecific gene silencing event which can block the ability of thatspecific gene to generate protein. In the nematode, this specific dsRNAcan be introduced simply by ingestion (Timmons and Fire, Nature,395:854, 1998; Timmons, Court and Fire, Gene, 263:103-112, 2001). IFTgenes are specifically targeted in the nematode by transforming planthosts so that they produce dsRNA derived from portions of genes encodingnematode IFT machinery, including kinesin-II and dynein 1b subunits aswell as the IFT raft proteins. This strategy allows for very specifictargeting of nematodes that attack and ingest part of the host plant.DNA/RNA sequences of portions of the IFT machinery will be chosen whichdo not have high homology to the homologous genes in other classes oforganisms so as to protect various animals and humans from potentialRNAi. Indeed, noncoding introns may be particularly useful for thispurpose.

[0317] Generating dsRNA via plant transformation with a stable vectorhas already been achieved in Arabidopsis (Chuang and Meyerowitz, ProcNatl Acad Sci, 97:4985-4990, 2000). For transformation, suitable vectorssuch as pCAMBIA vectors can be used. To generate the dsRNA, as little as100 base pairs of coding or noncoding sequence would be linked todouble-stranded complementary sequence so that when the RNA isgenerated, the two matching RNA sequences would base pair with oneanother and form double stranded RNA. Attacking nematodes would ingestthis dsRNA which would lead to RNA silencing of IFT genes which wouldresult in the loss of the sensory cilia. We would likely want to use astrong promoter so that the concentration of dsRNA in the plant cellswould be relatively high. The actual dose needed to affect loss ofciliary function would be measured in a laboratory setting. One positiveaspect of RNAi technology is that small amounts of RNA appear to beamplified within the nematode and transmitted adjacently (Fire et al.,Nature, 391:806-811, 1998).

[0318] IFT and Insect Pests

[0319] All insect pests utilize sensory cilia for important lifefunctions, including location of mates and identification of foodsources. Sensory cilia (ciliated sensory neurons) are the organelles forsmell, taste and hearing in insects. A compound that inhibited IFT wouldblock sensory cilia function and/or assembly, leaving an insect unableto smell, unable to taste, and unable to hear. Insects deprived of thesesenses would be unable to locate mates; as a result, their populationswould be controlled. Many insects deprived of these senses also would beunable to sense food, and thus would not eat, or would eat plants thatwould be harmful to them; this would both reduce insect populations andreduce damage to crop plants. Therefore, a compound that blocked IFTwould be a very effective pesticide. Such a compound can be applied byspraying of fields, plants, insect habitats, etc., or by any otherpreferred method.

[0320] For example, the male cabbage looper moth (Trichoplusia ni) usesciliated sensory neurons, called sensilla, which are located on itsantennae, to find a female moth (Borroni and O'Connell, J. Comp.Physiol., 170:691-700, 1992). The female moth releases a specificpheromone, which the male then detects by means of its sensilla andfollows upwind until it locates the female, with which it then mates. Bydisrupting the assembly and/or function of the male's sensilla, ananti-IFT compound would render the male moth unable to find the female,and the thus control the population of this major pest.

[0321] In another example, mosquitoes use olfactory receptors located onsensory cilia to detect CO₂ and other attractants, and thus find theirprey (Grant et al., J. Comp. Physiol., 177:389-396, 1995). Sprayingmosquitoes with an anti-IFT compound, which would block sensory ciliaassembly/function, would render the insect unable to locate its prey andobtain a blood meal.

[0322] Combating Insects

[0323] The approaches described above regarding nematodes can be adaptedto combat insects. Insects that attack and consume plants are goodtargets for plant transformants that generate either anti-IFT peptidesor double stranded RNA that is derived from insect genes encoding IFTproteins. Blocking IFT in the insect should result in at least twodesirable effects. First, the chordotonal organ, also known asJohnston's organ, contains a ciliated structure that is necessary forsound transduction. Second, insect sperm is flagellated and motile.Interruption of IFT should result in (1) deaf insects, which will affectcommunication with other insects, including potential mates and (2)immotile sperm, which will the males of the species infertile. Both ofthese effects can dramatically reduce the population of an insectspecies. Additional cells/tissues that can be affected by blocking IFTinclude the mechanosensory bristles. Many mutations that affect soundtransduction by the chordotonal organ also affect touch transduction bythese tactile bristles (Eberl, Hardy, and Keman, J. Neurosci.,20:5981-5988, 2000); these results suggest that IFT, or portions of theIFT machinery, is involved with touch transduction in insects. Losingthe sense of touch as a result of anti-IFT agent(s) would result in anobvious disadvantage for the organism.

EXAMPLES

[0324] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

Example 1 Identification of the Genes Encoding Chlamydomonas IFTParticle Proteins and Homologous Proteins in Other Species

[0325] Purification and Microsequencing of Chlamydomonas IFT ParticleProteins 16S IFT particles were purified from Chlamydomonas flagella asdescribed in Cole et al. (1998), further purified by two-dimensional gelelectrophoresis and transferred to ImmobilonP^(SQ) (Millipore Corp.,Bedford, Mass.). The spots corresponding to each of the IFT particleproteins were excised and digested with trypsin. Tryptic peptides wereeluted from the membrane and fractionated by high performance liquidchromatography. Pure peptides, identified by mass spectrometry, weresubjected to microsequence analysis in the UMMS Protein SequencingFacility.

[0326] Cloning Genes Encoding Chlamydomonas IFT Particle Proteins

[0327] The peptide sequences from the purified IFT particle proteinswere used to design PCR primers that were then used to amplify portionsof the genes from Chlamydomonas genomic DNA or cDNA. These fragments ofDNA were then used to obtain the rest of the genes by screening cDNAlibraries by DNA hybridization, or the fragments sequenced and thesequences used to design additional primers that were used in 5′ and 3′RACE (rapid amplification of cDNA ends) reactions to PCR amplify theremainder of the genes.

[0328] Obtaining Mouse IFT57 sequence

[0329] The predicted peptide from Chlamydomonas IFT57 was used to searchthe ESTS portion of Genbank. One positive ESTS clone (Accession#AA763980) was purchased from ATCC and sequenced.

[0330] Identifying Homologous Genes

[0331] The predicted peptide sequences of the Chlamydomonas IFT particleproteins were used to search the protein and ESTS portions of Genbankwith BLAST to identify homologous proteins. Typically human andChlamydomonas homologues were 30-50% identical at the protein sequencelevel. If a human homologue was not present in either the protein orESTS portion of Genbank, then the Chlamydomonas sequences (and anymammalian homologues found above) were used to search the draft humangenome sequence to identify homologous loci. The proteins predicted tobe encoded at these loci were compiled using the homology to theChlamydomonas (or non-human mammalian) homologue as a guide. Similarmethods were used to identify Caenorhabditis elegans homologues and canalso be used to identify genes encoding IFT particle proteins in anyorganism (e.g. Giardia, Plasmodium, Drosophilia) for which extensive DNAsequence data has been obtained.

Example 2 Correction of IFT Defects in Retinal Cells by Gene Therapy

[0332] Defects in IFT particle proteins cause retinal degeneration inmice (Pazour et al., 2001) and are likely to be a cause of degenerativeretinal disease leading to blindness in humans (Rosenbaum and Witman,2001). In such cases vision may be corrected by gene therapy methods inwhich a vector containing the wild-type gene for the defective IFTparticle protein is injected subretinally and the IFT particle proteingene thereby transfected into the photoreceptor cells and expressed.

[0333] The vector can be any non-viral or viral vector, e.g.,recombinant adeno-associated virus (rAAV) vector (Ackland et al., 2001),which has the ability to transfect the target retinal cells. Expressionof the IFT gene would be driven by its endogenous promoter, or by aphotoreceptor cell-specific promoter, e.g., the opsin promoter, or byanother promoter, such as the immediate-early cytomegalovirus (CMV)enhancer-promoter, which would be placed upstream of the IFT gene in theconstruct. Plasmid DNA containing this construct would be packaged intothe vector.

[0334] In the case of rAAV, this can be achieved by inserting the geneinto a basic rAAV vector plasmid, e.g., pTR-UF5, and transfecting theplasmid into a suitable human host cell in culture, e.g., human 293cells, together with helper plasmid required for productive AAVinfection and packaging of the rAAV DNA. After harvesting cells, thevirus is extracted by a standard procedure, e.g., by freezing andthawing the cells, and then purified by iodixanol density gradientcentrifugation followed by heparin-Sepharose agarose columnchromatography (Hauswirth et al., 2000 Meth. Enzymol. 316: 743-761).Alternatively, any other procedure which results in efficient recoveryof high quality virus may be used.

[0335] The purified vector would then be injected into the subretinalspace underlying the central retina by means of an anterior chambercannula inserted through a sclerotomy and monitored by microscopy(Bennett et al., 1999). Related protocols have been demonstrated to givestable, long-term expression of the transgene in non-human primatephotoreceptor cells (Bennett, J. et al., 1999. PNAS 96: 9920-9925),without toxicity, and to restore vision in a large animal model—thedog—in which retinal degeneration occurs as a result of a defect in theRPE65 gene (Acland et al., 2001).

Example 3 Correction of IFT Defects in Airway Epithelial Cells by GeneTherapy

[0336] Because IFT particle proteins are necessary for the formation andmaintenance of all cilia and flagella, defects in IFT particle proteinsare expected to prevent normal assembly and functioning of respiratorytract cilia. Defects in assembly and functioning of respiratory tractcilia lead to human disorders such as primary ciliary dyskinesia(Afzelius and Mossberg, 1995), which are characterized bybronchiectasis, chronic bronchitis, and chronic sinusitis. Respiratorytract cilia arise from epithelial cells, which are exposed on thesurface of the airway. Thus, these cells should be ideal targets forgene therapy to correct pulmonary disease due to a defect in an IFTparticle protein. In this case, viral or nonviral vectors containing theIFT gene can be delivered topically to the airways via direct liquidinstillation (Yonemitsu et al., 2000) or via an aerosol, such as can beproduced by a nebulizer (Gautam et al., 2001).

[0337] The wild-type IFT particle protein gene to be transfected intothe target cells can be contained in a plasmid (“IFT gene plasmid”) andcan be under the control of its endogenous promoter or another promoter,e.g., the human CMV early promoter/enhancer element. In one approach,the purified plasmid can be inserted into a rAAV vector plasmid forproduction of rAAV in a suitable human host cell, followed bypurification of the rAAV as described above for gene therapy methods tocorrect a defect in the retina. The purified rAAV can then be deliveredto the airway epithelial cells by instillation of a solution containingthe rAAV, or by aspiration of an aerosol containing the rAAV. Relatedapproaches might use recombinant lentiviral vectors (Kobinger et al.,2001) or recombinant Sendai virus vectors (Yonemitsu et al., 2000) totransfer the IFT gene to the airway epithelial cells. Both of thesevectors have been shown to efficiently transduce airway epithelial cells(Kobinger et al., 2001; Yonemitsu et al., 2000).

[0338] Alternatively, the IFT gene plasmid can be complexed with othercomponents to create a nonviral vector that can fuse with the airwayepithelial cell membrane and deliver the plasmid DNA to the cytosol,from where it would then move to the nucleus. For example, the IFT geneplasmid DNA can be mixed with cationic lipids to produce a DNA-cationiclipid complex or “liposome” that can be delivered as an aerosol or vialiquid instillation. Additional components, such as protamine orproteins, may be added to increase efficiency of delivery of the vectorto the target cell (e.g., Sorgi, FL et al., 1997. Gene Therapy4:961-968). Gene transfer efficiency is 10-200 times higher usingliposomes containing specific peptides—e.g., an integrin-bindingmotif—than using liposomes alone (Scott et al., 2001. J. Gene Med.3:125-134). In another approach, the DNA can be complexed withpolyethylenimine (PEI) to create a PEI-DNA complex. PEI-DNA complexeshave been demonstrated to be effective for the aerosol delivery ofreporter genes to the lungs of mice, and may be more effective thanliposomes (A. Gautam et al., 2000. Mol. Therapy 3:551-556).

[0339] With either viral or nonviral vectors, the efficiency of deliveryof the DNA to the target cells may be enhanced by directing the vectorto a specific target, e.g., the extracellular ATP/UTP receptor, termedP2Y2-R, which internalizes into the cell via clathrin-coated pits uponagonist stimulation (Boucher, RC. 1999. J. Clin Invest. 103:441-445).

Example 4 Determining the Role of IFT88 for Assembly of Cilia andFlagella

[0340] Introduction

[0341] Defects in the Tg737 gene cause kidney and liver defects in micethat are very similar to those seen in humans with autosomal recessivepolycystic kidney disease (ARPKD) (Moyer et al., 1994). This diseaseaffects—1 in 10,000 children born each year and may be responsible for amuch higher proportion of stillbirths and prenatal deaths (Blyth andOckenden, 1971; Cole et al., 1987). The function of the Tg737 protein isunknown. Here we identify a protein in Chlamydomonas that is homologousto Tg737 and show that it is required for assembly of flagella.

[0342] The epithelial cells lining the collecting tubules of the kidneyhave very well developed primary cilia (Andrews and Porter, 1974). Therole of these cilia is unknown; however they extend into the lumen ofthe tubule and may serve as sensory appendages. Precedence for primarycilia serving a sensory role is well established in vision andolfaction, as the outer segments of the rod and cone cells of the eyeand the olfactory cilia of the nose have evolved from cilia and haveretained primary cilia characteristics, e.g. the 9+0 microtubulearrangement. Primary cilia in other organisms such as Caenorhabditiselegans also serve a sensory role (Perkins et al., Dev. Biol.,117:456-487, 1986; White et. al., Phil. Trans. R. Soc. Lond, 275:327348,1976).

[0343] Eukaryotic cilia and flagella are built and maintained by aprocess called intraflagellar transport (IFT) (Rosenbaum et al., 1999).Most well characterized in Chlamydomonas, IFT is a rapid movement ofparticles along the axonemal microtubules of cilia and flagella. Theoutward movement of these particles from the cell body to the tip of theflagellum is driven by FLA10 kinesin-II (Kozminski et al., 1993; 1995),whereas the transport of the particles from the tip back to the cellbody is driven by DHC1b/DHC2 cytoplasmic dynein (Pazour et al., 1998,1999; Porter et al., 1999). The particles that are transported by IFTare composed of at least 17 protein subunits (Pipemo et al., 1997; Coleet al., 1998). The functions of the individual subunits are not knownbut the proteins are conserved between green algae, nematodes, andvertebrates (Cole et al., 1998; Rosenbaum et al. 1999). In thismanuscript, we describe the cloning of the IFT88 subunit of theChlamydomonas IFT particle and show that cells missing this gene do notassemble flagella. We further show that IFT88 is homologous to thepolycystic kidney disease gene Tg737 and that mice with mutations inthis gene have shorter than normal primary cilia in their kidney.

[0344] Materials And Methods

[0345] Purification and Microsequencing of Chlamydomonas IFT88

[0346] 16S IFT particles were purified from Chlamydomonas flagella asdescribed in Cole et al. (1998). The IFT88 subunit was further purifiedby two-dimensional gel electrophoresis and transferred toImmobilonP^(SQ) (Millipore Corp., Bedford, Mass.) as describedpreviously (Cole et al., 1998). The spot corresponding to IFT88 wasexcised and digested with trypsin. Tryptic peptides were eluted from themembrane and fractionated by high performance liquid chromatography.Pure peptides, identified by mass spectrometry, were subjected tomicrosequence analysis in the UMMS Protein Sequencing Facility.

[0347] Cloning IFT88

[0348] Portions of the IFT88 peptide sequence (LEGETDQA and GIDPYCVE)were used to design two degenerate oligonucleotide PCR primers (GA[A/G]AC[C/G/T] GA[C/T] CA[A/G] GC[C/G/T] GA[C/T] AA[A/G] TA and GC [C/T]TC[A/C/G]AC [A/G]CA [A/G]TA [A/C/G]GG [A/G]TC [A/G]AT). These primersamplified a 365-bp fragment of genomic DNA that contained parts of twoexons and a 132-bp intron. This fragment of genomic DNA was used toscreen a Chlamydomonas cDNA library made from cells undergoing division(Pazour and Witman, unpublished). Two positive clones were identifiedand sequenced by primer walking. These two clones were similar exceptfor the sequences at their 5′-ends. IFT88cDNA-1 was longer thanIFT88cDNA-2 and appeared to have a short region of poly-Ainappropriately fused to the 5′-end, probably the result of a cloningartifact. One Chlamydomonas IFT88 EST clone is in Genbank (accessionnumber AV395576). This EST sequence, which is from the 5′ end of thegene and overlaps the cDNA clones, was used to define the 5′-end of thecDNA sequence.

[0349] Four independent BAC clones (40-B3, 11-021, 24-F2, and 27-M3)were found in the Genome Systems (St Louis, Mich., USA) ChlamydomonasBAC library by Southern hybridization using the 365-bp fragment of IFT88genomic DNA as a probe. These four BAC clones were purchased from GenomeSystems. The presence of the IFT88 gene in the clones was confirmed bySouthern blotting.

[0350] Identification and Rescue of an IFT88 Mutant DNA from each of the˜400 transformants in our insertional mutant collection (Pazour et al.,1995; 1998; Pazour and Witrnan, 2000) was cut with PstI and analyzed bySouthern blotting with the 365-bp fragment IFT88 genomic DNA fragment.This probe detected an ˜2.5-kb band in wild-type cells and all of themutants except strain V79.

[0351] The motility/flagellar defect in V79 was rescued by transformingwith BAC clones carrying the IFT88 gene. Transformation was performed bythe glass bead method of Kindle (1990), and rescued cell lines wereidentified by restoration of their ability to swim. One rescued cellline was crossed to wild-type CC 124 cells. Tetrads from this cross weredissected and analyzed by standard procedures (Levine and Ebersold,1960; Harris, 1989) as described in Pazour et al. (1998). The flagellarphenotype was scored by light microscopy when the cells were in theearly log phase of growth.

[0352] Electron Microscopy

[0353] Chlamydomonas cells were fixed in glutaraldehyde for EM (Hoopsand Witman, 1983) and processed as described in Wilkerson et al. (1995).Tissues of anesthetized mice were fixed in situ by brief cardiacperfusion with 2.5% gluteraldehyde in 100 mM cacodylate buffer. Thekidneys were removed and a small amount of additional fixative wasinjected under the capsule of the kidney. The kidneys were placed inadditional fixative for 1 hour. At that time, the kidneys were sliced inhalf and further fixed for 2 days. The tissue was freeze fractured andmetal impregnated as described in McManus et al. (1993).

[0354] Western Blotting

[0355] Whole cell extracts of wild-type and mutant cells were made byresuspending log-phase cells in SDS-sample buffer, heating at 50° C. for10 min, and repeatedly drawing the sample through a 26-guage needle toshear the DNA. Proteins were separated by SDS-PAGE, blotted ontopolyvinylidene difluoride membranes, and probed with antibodies asdescribed in Pazour et al. (1998). Antibodies used included mAb57.1,mAb81.1, mAb139.1, and mAb172.1, which are monoclonal antibodies againstIFT particle proteins (Cole et al., 1998); FLA10N, which is specific fora kinesin-II motor subunit (Cole et al., 1998); DHC1b, which is specificfor the heavy chain of DHC1b/DHC2 cytoplasmic dynein; and B-5-1-2, whichis specific for alpha tubulin (Piperno et al., 1985).

[0356] Chlamydomonas Culture

[0357] Chlamydomonas strains used in this work included: g1 (nit1, agg1,mt+) (Pazour et al., 1995), CC124 (nit1, nit2, mt−), and CC1390 (fla2,mt−). The latter two strains are available from the ChlamydomonasGenetics Center (Duke University, Durham, N.C.). Strains generated inthe course of this work included: V79 (ift88-1::NIT1, mt+), which wasgenerated by random insertional mutagenesis of g1, V79/40-B3#2.5(ift88-1::NIT1, IFT88, mt+) generated by transformation of V79 with BACclone 40-B3, and 3276.2 (ift88-1 ::NIT1) which is a progeny from a crossbetween V79/40-B3#2.5 and CC124.

[0358] Mouse Genotyping

[0359] DNA was purified by digesting mouse tails with proteinase K,extracting once with 50% phenol/50% chloroform, once with chloroform andthen precipitating the DNA with ethanol. The genomic DNA was amplifiedusing the RW450, RW451, and RW452 primer set described by Yoder et al.(1997). These primers amplified a270-bp fragment from the wild-typelocus and a 340-bp fragment from the mutant locus.

[0360] Digital Image Processing

[0361] Western and Southern blots were scanned from negative x-ray filmwith a Linotype-Hell Saphir Ultra 2 flatbed scanner and brought intoPhotoshop for cropping and contrast adjustment. Scanning EM micrographswere scanned from positive Polaroid film in the same way. TransmissionEM negatives were scanned with a Polaroid Sprint Scan 45 and broughtinto Photoshop for cropping, contrast adjustment and inversion from anegative to a positive image.

[0362] Results

[0363] Cloning and Sequencing of Chlamydomonas IFT88

[0364] In order to learn more about the structure and function of theproteins that make up the IFT particle, we cloned and sequenced theIFT88 protein, formerly known as p88 (Cole et al., 1998). To do this,Chlamydomonas IFT particles were purified from the matrix of isolatedflagella by sucrose density gradient centrifugation and two-dimensionalgel electrophoresis. IFT88 was cleaved by trypsin and two internalpeptides were microsequenced (Cole et al., 1998), yielding the sequencesAATNLAFLYFLEGETDQADKYSEMALK and SLFNEAAGIDPYCVEAIYNLGLVSQR. DegeneratePCR primers were designed from these sequences and used to amplify afragment of genomic DNA. A cDNA library was screened with the genomicfragment and the resulting clones were sequenced by primer walking.Southern blots indicated that there is only one copy of the IFT88 genein the Chlamydomonas genome.

[0365] Sequence analysis showed that the IFT88 cDNA contains a 2346-ntopen reading frame that is predicted to encode an 86.3-kD protein with ap1 of 5.87. Perfect matches to both IFT88 tryptic peptides are found inthe open reading frame of this cDNA, rigorously confirming that theseclones encode the Chlamydomonas IFT88 protein. No discernable motifswere identified within the sequence except for the presence of 10tetratricopeptide repeats (TPR). TPRs are degenerate 34-amino acidrepeats (Lamb et al., 1995), present in tandem arrays of 3-16 units thatare predicted to form amphipathic helices (Hirano et al., 1990). Thefirst three TPR motifs are found closely spaced between amino-acidresidues 185-294. The other seven TPR motifs occur without spacingbetween amino-acid residues 441-676.

[0366] Chlamydomonas IFT88 Is Homologous to a Mouse Polycystic KidneyDisease Gene

[0367] Blast searches with the Chlamydomonas IFT88 protein sequenceindicate that it is very similar to the mouse (41% identical; BLASTE=e-148) and human Tg737 (40% identical; BLAST E=e-146) proteins. Micewith defects in Tg737 have severe polycystic kidney disease and diewithin a few weeks of birth. The protein also is homologous to proteinspredicted by ESTs from zebra fish and swine and fragments of preliminaryC. elegans and D. melanogaster genomic sequence. IFT88 and Tg737 arelikely to be functionally equivalent orthologues as the similaritybetween the Chlamydomonas and mammalian proteins is robust anddistributed over the entire coding region and not just within the TPRdomains. 40% identity is very typical of the amount of similarity seenbetween other Chlamydomonas and mammalian orthologues that encodeflagellar proteins (Pazour, Dickert and Witman, manuscript inpreparation).

[0368] IFT88 Is Required for Flagellar Assembly

[0369] To learn more about the function of IFT88 in cells, we searchedour collection of Chlamydomonas insertional mutants (Pazour et al.,1995, 1998; Pazour and Witman, 2000) for a cell line with a defect inthis gene. The insertional mutants were made by transforming cells withDNA carrying a selectable marker. In Chlamydomonas, transforming DNA isintegrated randomly throughout the genome and disrupts genes at the siteof integration. DNA was isolated from ˜400 insertional mutants havingbehavioral or motility defects, and was screened by Southern blottingusing a fragment of IFT88 genomic DNA as probe. One cell line (V79) wasidentified that had an insertion in the IFT8 gene. The fact that thesingle hybridizing band in wild-type cells was split into two bands inthe mutant indicated that the selectable marker integrated into the genewithin the region covered by the probe and did not result in a largedeletion of the genome at the site of integration. The mutant allele wastermed ift88-1.

[0370] The ift88-1 cells grew at the same rate as wild-type cells,indicating that IFT88 is not required for processes essential for growthor cell division. However, in contrast to wild-type cells that normallyhave two ˜10-μm flagella extending from the anterior end of the cellbody, the ift88- 1 cells completely lack flagella. Electron microscopicanalysis of these cells showed that the basal bodies were structurallynormal but the flagella did not extend beyond the transition zone. Insome cells, the membrane covering the flagellar tips was tightly apposedto the microtubules with no material between them and the membrane. Inother cells, the flagellar stubs were slightly swollen and containedfragments of microtubules in random orientations. However, in contrastto the IFT mutants fla14 (Pazour et al., 1998) and DHC1b (Pazour et al.,1999), no accumulation of IFT particles was observed in any of theflagellar stubs.

[0371] To determine the effect of the lack of IFT88 on the IFT particleand the IFT motors, we examined whole cell extracts by western blottingThe IFT particle is made up of two large complexes. Complex A iscomposed of four to five proteins and includes IFT139. Complex B iscomposed of IFT88 and ten other proteins including IFT172, IFT81 andIFT57. The complex A protein IFT139 is enriched in the mutant suggestingthat the gene may be upregulated in the mutant cells. The mutation haslittle or no effect on the levels of complex B proteins IFT172 andIFT81, but causes a significant decrease in IFT57, another complex Bprotein. The cellular levels of the IFT motors FLA10 kinesin-II andDHC1b are not affected by the ift88 mutation.

[0372] To be certain that the flagellar assembly defect is caused by themutation in IFT88 and is not the result of another mutation elsewhere inthe genome, we transformed the mutant cells with BAC clones carrying theIFT88 gene. Three independent BAC clones (40-B3, 24-F2, and 27M3)complemented the flagellar defect. The complemented cell lines swam likewild-type cells and had IFT. One of the rescued cell lines was crossedto a wild-type cell line and 26 tetrads were dissected. All fourproducts of one tetrad and a single product of the remaining 25 tetradswere analyzed by Southern blotting. Because the transformed copy of theIFT8 gene inserted at a site unlinked to the original locus, theinserted DNA segregated independently from the original gene, allowingoffspring to carry zero, one, or two copies of the wild-type gene. Cellsthat carry at least one copy of wild-type IFT88 have normal flagella andmotility, whereas those that carry no copies of wild-type IFT88 lackflagella and are non-motile. These data indicate that the flagellardefect is tightly linked to the ift88 mutation and is almost certainlythe result of it.

[0373] Primary Cilia in the Kidney of Tg737 Mice are Shorter Than Normal

[0374] Primary cilia are present on many cells in the mammalian body(Wheatley, 1995;

[0375] Wheatley et al., 1996), and are particularly well developed inthe kidney (Andrews and Porter, 1974). Inasmuch as Chlamydomonas IFT88is necessary for assembly of flagella and is homologous to mammalianTg737, and because a defect in mouse Tg737 leads to kidney disease, itwas of great interest to determine if the defect in Tg737 affectedformation of the primary cilia in the kidney. In wild-type rats, thecilia are ˜2.5 μm long and are found in the proximal tubule, the loop ofHenle, the distal tubules, and the collecting ducts (Andrews and Porter,1974). In wild-type mice these cilia are less than 5 μm long andsimilarly distributed (Flood and Totland, 1977).

[0376] We obtained the hypomorphic Tg737-mutant mice from Oak RidgeNational Laboratory and examined the kidneys of 4-day and 7-day old pupsby scanning electron microscopy. Numerous monociliated cells wereobserved in the kidneys of both wild-type (+/+) and homozygous mutant(−/−) mice, but the cilia in the mutant kidneys were much shorter. Toquantify this difference, the cilia lengths were measured from scanningelectron micrographs taken from the tubules distal to the proximaltubule. The proximal tubule was avoided because it contains a thickbrush border that can obscure a micron or more of cilia length. Thetubules distal to the proximal tubule have only sparse microvilli thatdo not obscure cilia, and the cilia in these regions are uniform inlength (Andrews and Porter, 1974). These cilia in wild-type mice were3.1+/−1.4 μm and 3.5+/−1.7 μm long at 4 and 7 days respectively, whereasthese cilia in the mutant mice were 1.0+/−0.6 μm and 1.3+/−0.6 μm at 4and 7 days respectively. These values represent minimum lengths as it isdifficult to accurately measure cilia that are lying at different anglesin the tubules. However, the differences are quite large and aresignificant at the >99% level. Thus, Tg737, like its IFT88 homologue inChlamydomonas, plays an essential role in assembly of the primary ciliumin the mouse.

[0377] The IFT88 Gene Is Required for Flagellar Assembly inChlamydomonas

[0378] Chlamydomonas cells lacking the IFT88 gene do not assembleflagella, indicating that the IFT88 protein is required for flagellarassembly. This is the first Chlamydomonas IFT particle subunit to beshown to be required for ciliary assembly. Loss of IFT88 causes asubstantial decrease in the amount of IFT57 relative to other IFTparticle proteins in the cytoplasm, suggesting that IFT88 is importantfor assembly of at least a portion of the IFT particle. Thus, IFT may beblocked at a very early stage in the ift88-1 mutant. Consistent withthis, IFT particles do not accumulate in the flagellar stubs of theift88 mutant, in sharp contrast to the dramatic accumulation ofapparently intact IFT particles in the flagella of mutants with defectsin cytoplasmic dynein DHC1b/DHC2 (Pazour et al, 1999) or the dyneinlight chain LC8 (Pazour et al., 1998). Alternatively, IFT88 may have avital role in the attachment of the IFT particle to its cargo or to theanterograde IFT motor FLA10-kinesin-II; in either case, loss of IFT88would preclude flagellar assembly. It is also possible that IFT88 isessential for transduction of a signal that is necessary for flagellarassembly.

[0379] Tg737, the Mouse IFT88 Homologue, is Required for Assembly of thePrimary Cilia in Kidney.

[0380] We have shown that IFT88 is highly similar to the mouse and humanTg737 proteins and that mice with defects in Tg737 have defective ciliain their kidneys. Tg737 was identified at Oak Ridge National Laboratoryby random insertional mutagenesis of mice. Hypomorphic mutations inTg737 cause kidney disease and death within a few weeks of birth. Thephenotype of this mutation closely resembles human ARPKD in that themice develop cystic kidneys and have hepatic biliary disease which isalso common in human patients with ARPKD (Moyer et al., 1994). The micedevelop large cysts in their collecting ducts and are unable toconcentrate urine (Yoder et al., 1996; 1997). Null alleles of Tg737 havea more severe phenotype and cause the mice to die during mid-gestation(Murcia et al., 2000). The phenotype caused by the null Tg737 mutationclosely resembles the phenotype of kinesin-II knockout mice (Nonaka etal., 1998; Marszalek et al., 1999, Takeda et al., 1999). Both thekinesin-II and Tg737 null mice have left-right asymmetry defects, lackcilia on the embryonic node, and die during mid gestation. Our findingthat IFT88 is required is required for ciliary assembly provides thefirst evidence that the lack of nodal cilia on embyros of Tg737 nullmutant mice is a direct result of a defect in IFT.

[0381] Primary cilia are extremely widely dispersed throughout themammalian body. The only cells that are known NOT to contain primarycilia are hepatocytes, and differentiated cells of myeloid or lymphoidorigin (Wheatley, 1995; Wheatley et al., 1996). The primary cilia inkidney tubules and ducts (Andrews and Porter, 1974) and hepatic biliaryducts (Motta and Fumagalli, 1974) are unusually long and project intothe lumens of these structures. The role of the primary cilia in thekidney or hepatic ducts is not known but has been suggested to besensory (Roth et al., 1988). The most studied primary cilia are theouter segments of rod and cone photoreceptor cells and the olfactorycilia in the nasal cavity. In these examples, the role of the primarycilia is clearly to serve as an appendage to concentrate sensorymachinery. C. elegans also makes extensive use of primary cilia todetect osmolarity gradients and chemical signals (White et. al., Phil.Trans. R. Soc. Lond., 275:327-348, 1976; Lewis and Hodgkin, Comp. Neur.,172:489-510, 1977). Primary cilia on other cells may similarly have asensory role. Supporting this idea, the somatostatin 3 receptor hasrecently been localized to primary cilia in the brain (Handel et al.,1999). Kidney epithelial cells sense multiple extracellular signalsincluding peptide hormones like angiotensin and ions like chloride(reviewed in Gunning et al., 1996). Whether any of these sensoryreceptors are localized to the primary cilia of the vertebrate kidneyremains to be determined.

[0382]C. elegans homologues of the human polycystic kidney diseasegenes, PKD 1 and PKD2, are localized to sensory cilia (Barr andStemberg, 1999). Humans with mutations in PKD1 and PKD2 develop kidneydisease similar to that caused by Tg737 mutations in mice. PKD1 and PKD2are transmembrane proteins that interact with each other (Qian et al.,1997; Tsiokas et al., 1997). PKD1 has a large extracellular domain thatis thought to bind an unknown ligand (Hughes et al., 1995; TheInternational Polycystic Kidney Disease Consortium, 1995). PKD2 ishomologous to calcium-regulated cation channels, suggesting that PKD2also is a cation channel (Chen et al., 1999). Further work will benecessary to determine if PKD1 or PKD2 are ever found on mammalianprimary cilia.

[0383] TPR Repeats in IFT88 and Tg737 Suggest That These Proteins areInvolved in Protein-Protein Interactions

[0384] TPR repeats are degenerate 34-amino acid motifs (Lamb et al.,1995) that are present in tandem arrays in proteins. These arrays arepredicted to form super-helices (Hirano et al., 1990) with amphipathicgrooves responsible for binding specific target proteins (Das et al.,1998). TPR domains have been found to mediate multiple simultaneousprotein-protein interactions in such multiprotein complexes as molecularchaperones and the anaphase-promoting complex 15 (reviewed in Blatch andLassle, 1999). In IFT88 and Tg737, there are three closely spaced TPRrepeats in the amino-terminal half of the protein and another seven TPRrepeats in the carboxyl-terminal half of the protein. These two separateTPR domains can serve to bind simultaneously to two separate targetproteins. These target proteins can be axonemal subunits that aretransported via IFT to the flagellar tip where they are assembled(Piperno et al., 1996). The targets also could be membrane proteins suchas receptors and channels, as IFT particles are tightly associated withthe flagellar membrane (Kozminski et al., 1995; Pazour et al., 1998).Alternatively, IFT88 c be binding to other subunits of the IFT particleand holding it together. IFT57 is likely to be an interacting proteinbecause it is destabilized in the absence of IFT88.

[0385] IFT is a Conserved Mechanism in the Assembly and Maintenance ofCilia and Flagella

[0386] A strong body of evidence indicates that IFT is necessary forassembly and maintenance of all eukaryotic motile and sensory cilia.Previous work has shown that the anterograde motor, kinesin-II, isnecessary for assembly and maintenance of cilia and flagella in diverseorganisms that include green algae, ciliated protozoa, nematodes,echinoderms, and vertebrates (reviewed in Cole, 1999; Marszalek andGoldstein, 2000). The retrograde motor, cytoplasmic dynein DHC1b/DHC2,also has been shown to be required for assembly of Chlamydomonasflagella (Pazour et al., 1999; Porter et al., 1999) and Caenorhabditissensory cilia (Signor et al., 1999; Wicks et al., 2000). Our initialreport on the composition of the Chlamydomonas IFT particle proteinsshowed that IFT52 was homologous to C. elegans OSM-6 and that IFT172 washomologous to C. elegans OSM-1 (Cole et al., 1998). OSM-1 and OSM-6 arerequired for assembly of sensory cilia in worms (Collet et al., 1998;Perkins et al., Dev. Biol., 117:456-487, 1986). The involvement of thesetwo nematode proteins in IFT was recently confirmed when GFP-labeledOSM-6 and OSM-1 were both shown to undergo IFT in transformed C. elegans(Orozco et al., 1999; Signor et al., 1999). The work in this paper showsthat the IFT particle protein IFT88 is required for ciliary assembly inChlamydomonas and that the IFT88 homologue, Tg737, is required forassembly of primary cilia in the kidney of mice. Thus, evidence from adiverse group of eukaryotes shows that both the IFT motors and the IFTparticle proteins are required for assembly and maintenance of cilia andflagella. This indicates that IFT is an ancient and conserved mechanismby which eukaryotic cilia and flagella are built and maintained.

[0387] IFT is Likely to Play Important Roles in Many Disease States

[0388] In addition to PKD discussed above, there are many other diseasesthat involve IFT. This includes retinitis pigmentosa (RP), which is agenetic disorder that causes destruction of photoreceptor cellsresulting in progressive vision loss. Transport of opsin and othercomponents of the rod outer segment is very important in photoreceptorcells, as 10% of the outer segment is turned over each day. Transportfrom the inner segment to the outer segment occurs through theconnecting cilium (reviewed in Besharse and Horst, 1990). Kinesin-II andseveral IFT particle proteins are located in the connecting cilium ofphotoreceptor cells (Beech et al., 1996, Whitehead et al., 1999; Pazouret al., submitted). Moreover, Marszalek et al. (2000) recently showedthat photoreceptor cells lacking kinesin-II accumulate opsin andarrestin in the inner segment, indicating that kinesin-II is involved intransport in photoreceptor cells. Therefore IFT is likely to be animportant transport mechanism in vertebrate photoreceptor cells, andmutations in IFT particle proteins are likely to cause vision defects.

[0389] Defects in IFT also are likely to affect motile cilia andflagella and be a cause of primary ciliary dyskinesia (PCD). PCD is asyndrome caused by defects in motile cilia and is characterized by maleinfertility, respiratory disease and situs inversus. It is well knownthat defects in axonemal components cause PCD (Afzelius, 1979; Pennarunet al., 1999). However, IFT particle proteins are highly expressed inthe testis and lung, suggesting that they are involved in the assemblyof motile sperm flagella and respiratory tract cilia (Pazour,unpublished). Thus, a mutation in an IFT particle protein can lead todefects in sperm flagellar assembly and result in sperm with shortdisorganized tails as have been described in some infertile human males(Baccetti et al., 1993). It is possible that mutations that preventassembly of both motile and sensory cilia are so severe that the embryosterminate during gestation, as has been observed with mutations in themouse kinesin-II motor subunits kif3a (Marszalek et al., 1999; Takeda etal., 1999) and kif3b (Nonaka et al., 1998).

Example 5 IFT Proteins in Vertebrate Photoreceptor Connecting Cilium

[0390] Introduction

[0391] Vertebrate photoreceptors are polarized sensory neuronsconsisting of a photosensitive outer segment, and an inner segment thatsupports synthesis of proteins destined for all cellular compartments.Transport of the visual pigment, opsin, and other components of thephototransduction machinery from the inner segment to the outer segmentis very important in photoreceptor cells, as ˜10% of the outer segmentis turned over each day (Young, J. Cell Biol. 33:61-72 (1967). Althoughdisruption of this process of intersegmental transport results inphotoreceptor degeneration and blindness, the mechanisms involved havenot been identified. Outer segments develop from a primary cilium whichremains as the sole connecting link and presumably the major transportcorridor between inner and outer segments (Besharse and Horst, InCiliary and Flagellar Membranes (ed Bloodgood, R. A.) pp. 389-417,Plenum Publishing Corp, New York, 1990). Recently, a process calledintraflagellar transport (IFT) has been found to be essential for theassembly and maintenance of sensory cilia in Caenorhabditis and themotile cilia of the green alga, Chlamydomonas, and sea urchins(Kozminski et al., J. Cell Biol. 131:1517-1527, 1995; Collet et al.,Genetics 148:187-200 1998; Morris et al., J. Cell Biol., 138:1009-1022,1997). During IFT, kinesin-II transports a large protein complex fromthe cell body to the tip of the flagellum (Piperno and Mead, Proc. Natl.Acad. Sci. USA, 94:4457-4462, 1997; Cole et al., J. Cell Biol.,141:993-1008, 1998; Orozco et al., Nature 398:674, 1999; Signor et al,Mol. Biol. Cell 10:345-360, 1999). These particles are then returned tothe cell body by the DHC1b/DHC2 form of cytoplasmic dynein (Pazour etal., J. Cell Biol, 141:979-992, 1998; Pazour et al., J. Cell Biol.,144:473-481, 1999; Porter et al., Mol. Biol. Cell, 10:693-712, 1999;Signor et al., J. Cell Biol., 147:519-530, 1999).). IFT particles arecomposed of 17 proteins and are thought to carry components needed forassembly and maintenance of cilia and flagella. They also transportsignals between cilia and the cell body (Rosenbaum et al., J. CellBiol., 144:385-388, 1999). The functions of individual IFT particleproteins are not known but mutations in genes encoding IFT particleproteins in Chlamydomonas and Caenorhabditis prevent ciliary assembly(Pazour et al., manuscript in preparation). IFT particle proteins areconserved among green algae, nematodes, and vertebrates. To investigatethe possibility that IFT functions in sensory cilia of vertebrates, wehave identified mouse homologues of three Chlamydomonas IFT particleproteins, have generated antibodies against the mouse proteins, andlocalized the proteins within the retina.

[0392] Cloning IFT20:

[0393] Chlamydomonas IFT20 was purified and the sequence of two trypticpeptides was obtained (GVYFDEDFHVR and YVSAIDQQVER) (Cole et al., J.Cell Biol. 141:993-1008, 1998). A degenerate PCR primer designed fromthe first peptide sequence was used in combination with an oligo-dTprimer to amplify most of the coding sequence from reverse-transcribedcDNA. The remainder of the gene was amplified from a Chlamydomonas cDNAlibrary in lambda ZapII (Stratagene) with a vector primer (M13Rev) and aIFT20-specific primer designed from the sequence of the first PCRproduct. The open reading frame contained within these clones encodes a15.6-kD peptide containing both tryptic peptides.

[0394] Bioinformatics:

[0395] Mouse and human homologues of the Chlamydomonas IFT particleproteins were identified by BLAST searches of Genbank. The human geneticmap positions were determined using publicly available data by thefollowing method. First, a mapped sequence tagged site (STS) from thegene itself (e.g. IFT20-1 and IFT52) or encoded within a genomic clonethat also encodes the IFT gene was identified in the STS portion ofGenbank. This allowed all the genes to be placed on the human radiationhybrid map. The approximate cytogenetic positions were then predictedusing the data of Bray-Ward et al. (Bray-Ward et al., Genomics 15:1-14,1996).

[0396] Antibody Production:

[0397] The antibodies were produced in rabbits by injecting bacteriallyexpressed maltose-binding fusion proteins made by cloning the openreading frames of mouse NDG5 (IFT52), IFT57, and IFT20 into the pMalcexpression vector (New England Biolabs, Mass.). The sera were affinitypurified using immobilized glutathione S-transferase fusion proteins.The latter fusion proteins were made by cloning the open reading framesof the IFT genes into the pGEX-6p-1 (Amersham Pharmacia Biotech, N.J.)expression vector.

[0398] Preparation of the Detergent Extracted Photoreceptor Cytoskeleton(DEPC) and Western Blotting:

[0399] The DEPC was prepared as described in Horst et al (Horst et al.,J. Cell Biol. 105:2973-2987, 1987). Briefly, rod outer segments shakenfrom 50 dark-adapted, frozen bovine retinas in Buffer A (10 mM PIPES, pH7.0, 1 mM EDTA, 5 mM MgCl₂, 0.02% NaN₃), supplemented with a proteaseinhibitor cocktail (1 μg/ml pepstatin, 1 μg/ml leupeptin, 4 μg/mlaprotinin, 1 mM benamidine, 1 mM PMSF) were purified by sucrose densitycentrifugation. Outer segments were then extracted in Buffer Acontaining 2% Triton X-100. Ciliary axonemes were separated fromdetergent-soluble material by centrifugation over a 45%-60% linearsucrose gradient. Equal aliquots were separated on a 12% denaturingpolyacrylamide gel and transferred to Immobilon membranes for antibodylabeling and detection using the SuperSignal West Femto chemiluminescentsystem (Pierce Chemical Co., IL).

[0400] Immunocytochemistry:

[0401] Fresh mouse or bovine retinal tissue was placed in TissueFreezing Medium™ (Triangle Biomedical Sciences, N.C.) and quickly frozenin liquid nitrogen with or without prior fixation in 4% paraformadehyde;Xenopus retina was prepared in the same way except that fixation was incold methanol (Whitehead et al., Exp. Eye Res. 69:491-503, 1999). Fishinner segment-outer segment preparations were made using proceduresdescribed in Beech et al. (Beech et al., J. Cell Sci., 109:889-897,1996). Primary antibodies were detected with goat anti-rabbit or goatanti-mouse IgG conjugated with Cy3 (Jackson Laboratories), Texas Red™,fluoroscein, Alexa 488, or Alexa 568 (Molecular Probes, Eugene Oreg.).In double-label experiments, discrimination of signals for K26 versusIFT proteins involved use of conjugated anti-mouse and anti-rabbitantibodies respectively. For discrimination of two monoclonal antibodies(K26 versus tubulin or opsin), we labeled with one monoclonal antibodyand a fluorescent anti-mouse antibody, and then repeated the procedurefor the second monoclonal antibody using a different fluorophore. Imagesof cells labeled with more than one fluorophore were pseudocolored andmerged using NIH Image 1.62 or Adobe Photoshop.

[0402] Identification of Vertebrate Homologues of Chliamydomonas IFTParticle Proteins

[0403] The Chlamydomonas IFT20, IFT52, and IFT57 particle proteins werepurified, partially sequenced, and the peptides compared to sequences inGenBank. Three peptides from IFT52 closely matched a rodent protein of˜52 kDa called NGD5, and a C. elegans protein called OSM6. The functionof NGD5 is unknown, but its expression is down-regulated by exposingcultured cells to opioids (Wick et al., Mol. Brain Res., 32:171-175,1995). OSM-6 is required for assembly of sensory cilia in nematodes. Thesequence of the Chlamydomonas peptides from IFT57 and IFT20 did not showstrong similarity to any proteins in the databases. Consequently,degenerate PCR was used to clone these two genes from Chlamydomonas. Thefull-length cDNA sequence of Chlamydomonas IFT57 strongly matched anumber of human and mouse EST sequences. To determine the extent ofhomology between the Chlamydomonas and mouse proteins, a mouse IFT57cDNA clone was completely sequenced. The Chlamydomonas and mouseproteins are 38% identical and have a BLAST p-value of 3e-67, indicatingthat the proteins are very likely to have related functions (Pazour etal., manuscript in preparation). The Chlamydomonas IFT20 sequencestrongly matched a small protein in mouse as well as EST sequences fromhumans, cattle and a large number of other vertebrate and invertebratespecies. The Chlamydomonas and mouse proteins are 32% identical and havea BLAST p-value of 4e-15. Bovine, mouse and human IFT20 proteins arenearly identical to each other.

[0404] We have obtained data indicating the chromosome locations of thethree human IFT protein homologues . Although none map to a regioncurrently associated with retinal degenerations, these data will proveuseful in future studies of photoreceptor disease or diseases resultingfrom defects in ciliary development. Analysis of genomic sequence dataindicated that in addition to the IFT20 sequence (IFT20-1) most similarto Chlamydomonas IFT20, at least two additional related genes arepresent in man; all three map to different chromosomes. All but one ofthe IFT20 EST sequences identified corresponded to IFT20-1. The ESTcorresponding to IFT20-2 has a stop codon within the region of homology,suggesting that it contains a sequencing error or does not encode afunctional protein.

[0405] Localization of IFT Particle Proteins to the VertebratePhotoreceptor:

[0406] Initial evidence for association of IFT particle proteins withphotoreceptor cilia came from western blot analysis. Bovine retinaltissue was used for this analysis in order to take advantage of aconnecting cilium-specific monoclonal antibody (K26) forimmunocytochemistry (Horst et al., Cell Motil. Cytoskeleton 17:329-344,1990) and a procedure for production of a detergent-extractedphotoreceptor cytoskeletal (DEPC) fraction from bovine retina (Horst etal., J. Cell Biol 105:2973-2987, 1987). Affinity-purified antibodies toIFT20, IFT52 and IFT57 did not readily detect proteins in whole cellextracts of bovine retina, but strongly detected single bands of ˜16, 52and 57 kDa respectively in the DEPC fraction. Because this fraction ishighly enriched in ciliary axonemes from photoreceptors, it seemed verylikely that the IFT proteins were associated with the photoreceptorcilia.

[0407] Immunocytochemical analysis revealed that IFT20, IFT52, and IFT57were most abundant in the inner segments (IS) of mouse (not shown) andbovine photoreceptors, with distinctly less labeling over the outersegments and other regions of the photoreceptor cells. The signal in theinner segment was distinctly granular in appearance, particularly at thejunction between the inner and outer segments where the connecting ciliaare located. The outer nuclear layer (ONL) containing photoreceptornuclei exhibited perinuclear staining. The inner nuclear layer (INL) wasalso labeled; the latter was most easily seen with antibodies to IFT52.All three antibodies labeled the outer plexiform layer which containsthe synaptic terminals of photoreceptors. This and the presence ofkinesin II in these synapses (Muresan et al., J. Neurosci.,19:1027-1037, 1999) suggests that IFT proteins have functions inphotoreceptor synaptic terminals.

[0408] Double-label immunocytochemistry with a monoclonal antibody (K26)that recognizes a connecting cilium specific epitope demonstrated thatall three IFT proteins are associated with the ciliary axoneme in situ.The K26 antibody uniquely stained the connecting cilium at the base ofphotoreceptor outer segments, which were identified with antibodies torod opsin. In contrast, antibodies to acetylated alpha-tubulin labeledmicrotubules of the inner segment and the ciliary axoneme. Labeling byboth K26 (red) and acetylated alpha tubulin (green) antibodies in theconnecting cilium resulted in a yellow to orange color due to overlap inthe connecting cilium, and demonstrated that axonemal microtubulesextend distally beyond the connecting cilium into the outer segment.Antibodies to IFT20, IFT52 and IFT57 labeled structures both on theproximal (inner segment) and distal (outer segment) side of theconnecting cilium in a large proportion of the photoreceptors. Frequentyellow to orange coloration of the connecting cilium was indicative ofoverlap of the two labels (red, K26 and green, anti-IFT antibody) withinthe connecting cilium. Triple-labeled images revealed a similar patternin which IFT57 (blue) is found in association with microtubules(acetylated α-tubulin) at both ends of the connecting cilium (K26) inmost but not all photoreceptors.

[0409] Association of IFT proteins with ciliary axonemes was alsodetected in the large rod photoreceptors of the frog, Xenopus laevis,and the fish, Lepomis macrochirus. Confocal analysis of Xenopus tissuesections double labeled with antibodies to acetylated α-tubulin andIFT57 revealed a single area of intense immunoreactivity in each cellcorresponding to the base of the ciliary axoneme, in the region of thebasal body. A similar pattern was seen in whole-mounted isolated cellsof Lepomis, although the zone of staining at the base of the axoneme wasbroader than in Xenopus. IFT57 staining of the distal axoneme within theouter segment was sometimes seen as punctate spots in both Xenopus andLepomis. These staining patterns are similar to those seen inChlamydomonas. In Chlamydomonas, the IFT particle proteins are locatedprimarily in a cytoplasmic pool at the base of the cilia with only asmall number of punctate spots found along the length of the cilia. Thepunctate staining is thought to be due to IFT particles that were intransit when the cells were fixed.

[0410] Discussion:

[0411] Macromolecules of the photoreceptor outer segment are synthesizedin the inner segment and transported into the outer segment. Thisprocess occurs at a prodigious rate. It has been estimated that turnoverin each mammalian photoreceptor outer segment requires delivery of asmany as 2000 photopigment molecules per minute throughout the life ofthe cell. In the larger photoreceptors of amphibians, this rate isincreased by more than an order of magnitude. In addition to thephotopigment molecules, proteins of the phototransduction machinery(Philp et al., FEBS Lett., 225:127-132, 1987; Whelan et al., J.Neurosci. Res. 20:263-270, 1988) and phospholipid components of thediscs (Anderson et al., Biochim. Biophys. Acta, 620:212-226, 1980) turnover rapidly. Although transport between the inner and outer segment iscrucial to polarized organization of the cell, the underlying mechanismhas remained elusive.

[0412] The presence of IFT proteins in photoreceptor cilia stronglysuggests that IFT is an important transport mechanism in these cells.Although the transported cargo has not been identified, our data allowus to propose the following model based on the idea that membranecomponents, including rhodopsin and phospholipid, and many solubleproteins are normally targeted to the photoreceptor outer segment.Membrane proteins are synthesized on the endoplasmic reticulum andmodified during passage through the Golgi network. The polarity of innersegment microtubules with their minus ends associated with the base ofthe cilium suggests that these membrane proteins and phospholipids aretransported from the Golgi stack to the base of the connecting cilium bydynein complexes containing the DHC1 heavy chain. DHC1 is a wellestablished vesicle transporter and has been shown to interact with thecytoplasmic tail of rhodopsin (Tai et al., Cell 97:877-887, 1999).Cytoplasmic proteins destined for the outer segment such as componentsof the transduction machinery (transducin and arrestin) and the ciliaryaxoneme are also synthesized in the inner segment and can be transportedin association with IFT particles. At the base of the connecting ciliumwhere IFT proteins are normally most concentrated, they associate withthe surface of these vesicles and with other outer segment proteins.Once the vesicles fuse with the plasma membrane adjacent to the cilium,transport of the complex with attached cargo along the connecting ciliumwould occur by the kinesin-II motor. Kinesin-II is thought to be theanterograde IFT motor in Chlamydomonas and Caenorhabditis and has beenlocalized to the connecting cilium in vertebrates (Beech et al., J. CellSci., 109:889-897, 1996; Whitehead et al., Exp. Eye Res., 69:491-503,1999). In the outer segment, the IFT particles disengage from theircargo, and the membrane is organized into disks and pinched off bymyosin VIIA. Myosin VII is required for phagocytosis in Dictyostelium(Titus, Curr Biol., 9:1297-1303, 1999), and mice with defects in themyosin VIIA gene accumulate opsin in the connecting cilium (Liu et al.,J. Neurosci. 19:6267-6274, 1999). Soluble proteins of the transductionmachinery and cytoskeleton would be expected to associate withappropriate protein complexes within the outer segment, while IFTparticles at the distal end of the connecting cilium would be returnedto the base of the cilium by a dynein complex containing the DHC1b/DHC2heavy chain. DHC1b/DHC2 is the retrograde IFT motor inChlamydomonas¹⁰⁻¹² and Caenorhabditis (Signor, J. Cell Biol.,147:519-530, 1999) and has been localized to the connecting cilium ofvertebrate photoreceptors (Besharse et al., in preparation). At the baseof the connecting cilium the IFT particles re-enter a peri-basal bodypool of IFT particle proteins and begin the cycle again. The IFTparticles also move components of the transduction machinery from theouter segment to the inner segment. For example, both transducin andarrestin have been shown to rapidly move between the segments duringlight and dark adaptation.

[0413] In conclusion, we have shown that three different IFT particleproteins are localized to the connecting cilium of vertebratephotoreceptor cells. Since IFT is essential for assembly and maintenanceof motile and sensory cilia of Chlamydomonas and Caenorhabditis, it islikely that it also is important in vertebrate photoreceptors. Theavailability of mouse mutations in the genes encoding kinesin-II(Nonaka, Cell 95:829-837, 1998; Bray-Ward et al., Genomics, 15:114,1996) and an IFT particle protein (Pazour et al., manuscript inpreparation) will allow us to directly test this hypothesis.

OTHER EMBODIMENTS

[0414] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

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What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a) a nucleic acid molecule having a nucleotidesequence which is at least 90% identical to the nucleotide sequence ofChlamydomonas intraflagellar transport (IFT) particle protein gene 20,27, 46, 52, 57, 72, 88, 122, 139, or Che-2, or a complement thereof; b)a nucleic acid molecule comprising at least 15 nucleotide residues andhaving a nucleotide sequence identical to at least 15 consecutivenucleotide residues of the nucleotide sequence of Chlamydomonas IFTparticle protein gene 20, 27, 46, 52, 57, 72, 88, 122, or 139, or Che-2,or a complement thereof; c) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of Chlamydomonas IFTparticle protein 20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2; or d) anucleic acid molecule which encodes a polypeptide comprising at least 10amino acids and having an amino acid sequence identical to at least 10consecutive amino acids of the amino acid sequence of Chlamydomonas IFTparticle protein 20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2.
 2. Theisolated nucleic acid molecule of claim 1, which is selected from thegroup consisting of: a) a nucleic acid having the nucleotide sequence ofChlamydomonas IFT particle protein gene 20, 27, 46, 52, 57, 72, 88, 122,139, or Che-2, or a complement thereof; and b) a nucleic acid moleculewhich encodes a polypeptide having the amino acid sequence ofChlamydomonas IFT particle protein 20, 27, 46, 52, 57, 72, 88, 122, 139,or Che-2.
 3. The nucleic acid molecule of claim 1, further comprisingnucleic acid sequences encoding a heterologous polypeptide.
 4. A vectorcomprising the nucleic acid molecule of claim
 1. 5. A host cellcomprising the nucleic acid molecule of claim
 1. 6. The host cell ofclaim 5, wherein the host cell is a non-human mammalian host cell.
 7. Anisolated polypeptide selected from the group consisting of: a) apolypeptide comprising at least 10 amino acids and having an amino acidsequence identical to at least 10 consecutive amino acids of the aminoacid sequence of Chlamydomonas intraflagellar transport (IFT) particleprotein 20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2; b) a polypeptidecomprising the amino acid sequence of Chlamydomonas IFT particle protein20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2, wherein the polypeptidecomprises one or more conservative amino acid substitutions that do notinhibit the biological activity of the polypeptide relative to acorresponding native Chlamydomonas IFT particle protein; and c) apolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 90% identical to a nucleic acidconsisting of the nucleotide sequence of Chlamydomonas IFT particleprotein gene 20, 27, 46, 52, 57, 72, 88, 122, 139, or Che-2, or acomplement thereof.
 8. The isolated polypeptide of claim 7, comprisingthe amino acid sequence of Chlamydomonas IFT particle protein 20, 27,46, 52, 57, 72, 88, 122, 139, or Che-2.
 9. The polypeptide of claim 7,wherein the polypeptide further comprises heterologous amino acidresidues.
 10. An antibody that selectively binds to the polypeptide ofclaim
 7. 11. An antibody that selectively binds to the polypeptide ofclaim
 8. 12. An isolated nucleic acid molecule selected from the groupconsisting of: a) a nucleic acid molecule having a nucleotide sequencewhich is at least 90% identical to the nucleotide sequence of mouseintraflagellar transport (IFT) particle protein gene 57, or a complementthereof; b) a nucleic acid molecule comprising at least 15 nucleotideresidues and having a nucleotide sequence identical to at least 15consecutive nucleotide residues of the nucleotide sequence of mouse IFTparticle protein gene 57, or a complement thereof; c) a nucleic acidmolecule which encodes a polypeptide comprising the amino acid sequenceof mouse IFT particle protein 57; or d) a nucleic acid molecule whichencodes a polypeptide comprising at least 10 amino acids and having anamino acid sequence identical to at least 10 consecutive amino acids ofthe amino acid sequence of mouse IFT particle protein
 57. 13. Theisolated nucleic acid molecule of claim 12, which is selected from thegroup consisting of: a) a nucleic acid having the nucleotide sequence ofmouse IFT particle protein gene 57 or a complement thereof; and b) anucleic acid molecule which encodes a polypeptide having the amino acidsequence of mouse IFT particle protein
 57. 14. An isolated polypeptideselected from the group consisting of: a) a polypeptide comprising atleast 10 amino acids and having an amino acid sequence identical to atleast 10 consecutive amino acids of the amino acid sequence of mouseintraflagellar transport (IFT) particle protein 57; b) a polypeptidecomprising the amino acid sequence of mouse IFT particle protein 57,wherein the polypeptide comprises one or more conservative amino acidsubstitutions that do not inhibit the biological activity of thepolypeptide relative to native mouse IFT particle protein 57; and c) apolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 90% identical to a nucleic acidconsisting of the nucleotide sequence of mouse IFT particle protein gene57, or a complement thereof.
 15. The isolated polypeptide of claim 14,comprising the amino acid sequence of mouse IFT particle protein
 57. 16.A method for identifying a candidate compound that modulates theactivity of mouse intraflagellar transport (IFT) particle protein 57,the method comprising: contacting a test compound to an isolated IFTparticle polypeptide of claim 14; and determining whether the testcompound interacts with the polypeptide, wherein interaction indicatesthat the test compound is a candidate modulator of mouse IFT particle 97protein
 57. 17. A method for identifying a candidate compound thatmodulates the activity of a human intraflagellar transport (IFT)particle protein, the method comprising: contacting a test compound toan isolated IFT particle polypeptide; and determining whether the testcompound interacts with the polypeptide, wherein interaction indicatesthat the test compound is a candidate modulator of a human IFT particleprotein.
 18. The method of claim 17, wherein the isolated human IFTparticle polypeptide is selected from the group consisting of human IFTparticle polypeptide 20-1, 20-2, 20-3, 27, 46, 52, 57-1, 57-2, 72, 88,122, 139-1, 139-2 and Che-2.
 19. The method of claim 17, wherein thetest compound binds to the isolated IFT particle polypeptide and whereinthe modulation is inhibition of activity.
 20. The method of claim 17,wherein the test compound binds to the isolated IFT particle polypeptideand wherein the modulation is increasing activity.
 21. The method ofclaim 17, further comprising contacting the candidate modulator to aculture of cells comprising functional cilia, and determining whetherthe candidate modulator inhibits cilia function, wherein inhibition ofcilia function indicates the candidate modulator is an IFT particleprotein inhibitory agent.
 22. The method of claim 17, further comprisingcontacting the candidate modulator to a culture of cells comprisingnon-functional cilia and lacking a specific IFT particle protein, anddetermining whether the candidate modulator restores cilia function,wherein restoration of cilia function indicates the candidate modulatoris an IFT particle protein restorative agent.
 23. A method foridentifying a candidate compound that restores the activity of adefective or absent human intraflagellar transport (IFT) particleprotein, the method comprising: obtaining a mixture of isolated IFTparticle polypeptides that comprises (i) all but one of the IFT particlepolypeptides required to form the IFT particle, and (ii) a medium thatenables the IFT particle polypeptides to form the IFT particle when allnormal IFT particle polypeptides that constitue that IFT particle arepresent; contacting a test compound to the mixture; and determiningwhether the test compound enables the IFT particle to be formed, whereinIFT particle formation indicates the test compound is a candidatecompound that restores the activity of a defective or absent human IFTparticle protein.
 24. The method of claim 23, further comprisingcontacting the candidate compound to a culture of cells comprisingnon-functional cilia and lacking a specific IFT particle protein, anddetermining whether the candidate compound restores cilia function,wherein restoration of cilia function indicates the candidate compoundis an IFT particle protein restorative agent.
 25. The method of claim23, wherein the human IFT particle polypeptide is selected from thegroup consisting of human IFT particle polypeptides 20-1, 20-2, 20-3,27, 46, 52, 57-1, 57-2, 72, 88, 122, 139-1, 139-2 and Che-2.
 26. Amethod of diagnosing a disorder in a tissue in a subject caused by adefective or absent human intraflagellar transport (IFT) particleprotein, the method comprising obtaining a sample of cells from thetissue; disrupting the cells; contacting the disrupted cell sample withan antibody that specifically binds to a normal human IFT particleprotein; and detecting binding of the antibody to any IFT particleprotein in the sample, wherein absence of binding indicates that thetissue has a disorder caused by a defective or absent IFT particleprotein.
 27. The method of claim 26, wherein the disorder is kidneydisease, retinal disorder, thyroid disorder, chondrocyte disease,olfactory disease, azoospermia, or primary ciliary dyskinesia.
 28. Amethod of treating a disorder in a subject caused by a defective orabsent intraflagellar transport (IFT) protein, the method comprisingadministering to the subject a human IFT particle polypeptide in anamount effective to restore the function of the defective or absent IFTparticle protein.
 29. The method of claim 28, wherein administering thehuman IFT particle polypeptide comprises administering a nucleic acidthat encodes a human IFT particle polyptide.
 30. The method of claim 28,wherein the human IFT particle polypeptide is selected from the groupconsisting of human IFT particle polypeptides 20-1, 20-2, 20-3, 27, 46,52, 57-1, 57-2, 72, 88, 122, 139-1, 139-2 and Che-2.
 31. A method oftreating an infection in a subject caused by a pathogen that comprises aintraflagellar transport (IFT) particle protein, the method comprisingadministering to the subject an effective amount of an agent thatinhibits the function of the IFT particle protein.
 32. The method ofclaim 31, wherein the agent is an antibody that binds specifically tothe IFT particle protein.
 33. The method of claim 31, wherein thesubject is a mammal.
 34. The method of claim 31, wherein the subject isa human.
 35. The method of claim 31, wherein the subject is a plant. 36.The method of claim 31, wherein the pathogen is a nematode, insect,protozoa bacteria.